Title of Invention

"A NUCLEIC ACID DIRECTING THE EXPRESSION OF A PROTEIN OF INTEREST IN THE CELLS OF THE POSTERIOR SERICIGENIC GLANDS OF BOMBYX MORI"

Abstract A nucleic acid directing the expression of a protein of interest specifically in the cells of the posterior sericigenic glands of Bombyx mori, wherein said nucleic acid comprises, from the 5' terminus to the 3' terminus, (i) a regulatory region comprising regulatory signals for the expression of a polynucleotide of interest specifically in said sericigenic cells and (ii) a region coding for a modified signal peptide placed under the control of said regulatory region, said nucleic acid having at least 90 % identity in nucleotides with the polynucleotide running from nucleotide 1150 to nucleotide 2026 of the sequence SEQ ID NO: 1 and in which the trinucleotide running from the nucleotide 1486 to nucleotide 1488 of sequence SEQ ID NO: 1 codes for an amino acid chosen from the group comprising alanine, isoleucine and leucine.
Full Text NUCLEIC ACID DIRECTING THE EXPRESSION OF A POLYPEPTIDE OF INTEREST IN THE POSTERIOR SERICIGENIC GLANDS OF A LEPIDOPTERAN
AND ITS APPLICATIONS
The invention relates to the field of synthesis and secretion of proteins of industrial interest, such as the fibrous proteins for using in textiles, in particular the novel silks or proteins for biomedical use. It relates to the targeted expression of polynucleotides and / or polypeptides of interest specifically in certain animal tissues and more precisely in the sericigenic gland of Bombyx mori. According to the invention, the sericigenic gland of the silk work is used as a "bioreactor" capable of producing large quantities of protein and exporting them into the silk cocoon from which they can be purified.
PRIOR ART
There exist methods for producing fibrous proteins for use in textiles such as the method for spider silk filament protein production by the mammary glands of transgenic goats (Nexis Biotechnologies, Inc. 1000 St-Charles Avenue, Bloc B, Vaudreuil-Dorion, QC, J7V 8PV, Canada). However, these methods do not allow organization of the fibrous protein in the form of a thread. As regards the production of soluble proteins for biomedical use, the known proteins imply bacterial cultures, cultures of cell lines, or vertebrate animals such as cows, goats or even transgenic rabbits. This type of production of recombinant proteins is very expensive. On the other hand, when the recombinant proteins are synthesized using vertebrate systems, their industrial utilization, especially their food - stuff or medical utilization, necessarily involves verification in advance that these proteins or the compositions containing them are free of pathogenic contaminants that can be passed on to the human being.
Given the important economic role of recombinant protein production, there is a need in the state of technology for new methods for producing recombinant proteins in large quantities at low costs and using an organism that is sufficiently remote from the human species to avoid problems of contamination, primarily when the protein is of biomedical interest. There is also a need for-new methods for producing recombinant proteins that make it possible to organize the protein in the form of a thread when said recombinant protein is fibrous. In order that the production can be high, the protein must be synthesized specifically in a tissue and exported to the exterior of the animal, preferably an invertebrate.
However, as far as the applicant knows, no invertebrate is capable of responding to the aforementioned requirements. The transgenesis of Bombyx mori, for example, is well known (Tamara et al. Germline transformation of the silkworm Bombyx mori L.

using a piggyBac transposon derived vector. Nature Biotechnology Vol. 18, 2000), but the utilization of this method has not yet made it possible to result in the production of recombinant proteins in large quantities.
The applicant has therefore dedicated efforts to developing a system of expression making it possible in particular to overcome the drawbacks of innocuousness and high costs of the previously known systems.
SUMMARY OF THE INVENTION
This type of system of expression of recombinant proteins is now provided by the invention.
The invention relates to a nucleic acid directing the expression of a protein of interest specifically in the cells of the posterior sericigenic glands of Bombyx mori, wherein said nucleic acid comprises at the 5' terminus to the 3' terminus, (i) a regulatory region comprising regulatory signals for the expression of a polynucleotide of interest specifically in said sericigenic cells and (ii) a region coding for a modified signal peptide placed under the control of said regulatory region, said nucleic acid having at least 90 % identity in nucleotides with the polynucleotide running from nucleotide 1150 to nucleotide 2026 of sequence SEQ ID N° 1 and in which the trinucleotide running from the nucleotide 1486 to nucleotide 1488 of sequence SEQ ID №1 codes for an amino acid chosen from the group comprising alanine, isoleucine and leucine.
The invention also relates to an expression cassette comprising a polynucleotide of interest placed under the control of a nucleic acid as defined hereinbefore, the polynucleotide of interest being a sense polynucleotide coding for a polypeptide.
The invention also relates to a recombinant integration vector comprising a nucleic acid or an expression cassette as defined hereinbefore as well as a host cell, modified by the integration of said nucleic acid or said expression cassette in its genome. Further still, the object of the invention is the use of a nucleic acid, an expression cassette, a recombinant vector or a recombinant host cell as defined hereinbefore for obtaining a transgenic non-human animal.
The invention also relates to processes for obtaining a transgenic non-human animal modified by integration into its genome of a nucleic acid, an expression cassette, a recombinant vector or a recombinant host cell as defined hereinbefore.
The invention also relates to a nucleic acid directing the expression of an RNA of interest reducing the synthesis of fibroin H, specifically in the cells of the posterior sericigenic glands of Bombyx mori, said nucleic acid comprising a regulatory region comprising regulatory signals for the expression of a polynucleotide of interest specifically in said sericigenic cells, said nucleic acid having at least 90 % nucleotide identity with the polynucleotide running from nucleotide 1150 to nucleotide 1452 of

sequence SEQ ID № 1.
A nucleic acid such as the aforementioned nucleic acid will be described in greater detail in the part entitled "Supplemental nucleic acids according to the invention."
DETAILED DESCRIPTION OF THE INVENTION
It has been shown, according to the invention, that a nucleic acid that comprises (i) a regulatory region comprising at least one copy of the target nucleotide motif of the transcription factor of the SGF1 "forkhead" type, an appropriate transcription promoter site and (ii) a region coding for a modified signal peptide, said region being placed under the control of said regulatory region, is capable of directing the synthesis of a protein of interest specifically in the posterior sericigenic cells of the lepidopteran Bombyx mori, when the nucleotide sequence coding for said protein of interest is fused "in phase" with the nucleotide sequence coding for said modified signal peptide.
According to the invention, the nucleotide sequence coding for the protein of interest is fused "in phase" with the nucleotide sequence coding for said modified signal peptide when the nucleotide localized at the 3' terminus of the last codon of the sequence coding for the modified signal peptide precedes the nucleotide located at the 5' terminus of the first codon of the sequence coding for the protein of interest.
Surprisingly, the applicant has shown that the replacement of the codon for cysteine of the signal peptide included in the pre-pro-protein fibrohexamerin of the silk thread fiber with a hydrophobic amino acid such as alanine, isoleucine or even leucine enables a protein fused with said modified signal peptide to be exported to the outside of the sericigenic glands of Bombyx mori, as constituent proteins of the silk thread.
The object of the invention relates to a nucleic acid directing the expression of a protein of interest specifically in the cells of the posterior sericigenic glands of Bombyx mori, wherein said nucleic acid comprises from the 5' terminus to the 3' terminus, (i) a regulatory region comprising the regulatory signals for the expression of a polynucleotide of interest specifically in said sericigenic cells and (ii) a region coding for a modified signal peptide placed under the control of said regulatory region, said nucleic acid having at least 90 % identity in nucleotides with the polynucleotide running from nucleotide 1150 to nucleotide 2026 of sequence SEQ ID N° 1 and in which the trinucleotide running from the nucleotide 1486 to nucleotide 1488 of sequence SEQ № 1 codes for an amino acid chosen from the group comprising alanine, isoleucine and leucine. Preferably, any nucleic acid and any polypeptide according to the invention are present in an isolated or purified form.
The term "isolated" in the meaning of the present invention is defined as a biological material that has been extracted from its original environment (the environment in which it is naturally found). For example, a polynucleotide present in

the natural state in an organism is not isolated. The same polynucleotide separated from the adjacent nucleic acids into which it is naturally inserted in the genome of the organism is isolated. This type of polynucleotide can be included in a vector and / or such a polynucleotide can be included in a composition and nevertheless remain in the isolate state, due to the fact that the vector or the composition does not constitute its natural environment. The same reasoning also applies to a polypeptide extracted from its natural environment.
The term "purified" does not require that the material will be present in an absolutely pure form, free of the presence of other compounds. It is a relative definition. A polynucleotide or a polypeptide is in a purified state after purification of the starting material or the natural material of at least one order of magnitude of preferably two or three and preferably four or five orders of magnitude.
For the purposes of the present invention, the expression "nucleotide sequence" can be used to designate indifferently a polynucleotide or a nucleic acid. The expression "nucleotide sequence" encompasses the genetic material itself and is thus not restricted to the information relating to its sequence. The terms "nucleic acid", "polynucleotide", "oligonucleotide", or even "nucleotide sequence" encompass the DNA, RNA, cDNA sequences or even the hybrid RNA / DNA sequences of more than one nucleotide, regardless of whether they are in single - stranded form or in duplex form.
The term "nucleotide" designates the natural nucleotides (A, T, G, C and U). For the purposes of the present invention, a first polynucleotide is considered to be "complementary" to a second nucleotide when each base of the first nucleotide is paired with the complementary base of the second polynucleotide, whose orientation is reversed. The complementary "bases" are A and T (or A and U), and C and G.
According to the invention, a first nucleic acid having at least 90 % identity with a second nucleic acid of reference will have at least 90 %, preferably at least 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 97.5 %, 98 %, 98.3 %, 98.6 %, 99 %, 99.6 % identity in amino acids with said second nucleic acid of reference. The "percent identity" between two nucleic acid sequences in the meaning of the present invention is determined by comparing the two sequences in optimum alignment and viewed through a comparison window.
The part of the nucleotide sequence in the comparison window can thus comprise additions or deletions (for example, "gaps") relative to the reference sequence (which does not comprise these additions or deletions) in order to obtain optimum alignment between the two sequences. The percent of identity is calculated by determining the number of positions at which an identical nucleic base is observed for the two sequences being compared, then by dividing the number of positions at

which there is identity between the two nucleic bases by the total number of positions in the comparison window, then multiplying the result by 100 in order to obtain the percent identity in nucleotides between the two sequences.
The optimum alignment of the sequences for the comparison can be done using computer means by means of well-known algorithms.
In a more preferred manner, the percent identity of the sequence is determined using the CLUSTAL W software (version 1.82) with the following settings : (1) CPU MODE = ClustalW mp ; (2) ALIGNMENT = « full» ; (3) OUTPUT FORMAT = « aln w/numbers » ; (4) OUTPUT ORDER = « aligned » ; (5) COLOR ALIGNMENT = « no » ; (6) KTUP (word size) = « default» ; (7) WINDOW LENGTH = « default» ; (8) SCORE TYPE = « percent» ; (9) TOPDIAG = « default» ; (10) PAIRGAP = « default »;(!!) PHYLOGENETIC TREE/TREE TYPE = « none » ; (12) MATRIX = « default » ; (13) GAP OPEN = « default » ; (14) END GAPS - « default » ; (15) GAP EXTENSION « default » ; (16) GAP DISTANCES - « default » ; (17) TREE TYPE = « cladogram » and (18) TREE RAP DISTANCES = « hide ».
A nucleic acid having at least 90 % nucleotide identity with a nucleic acid according to the invention encompasses the "variants" of a nucleic acid according to the invention.
A "variant" of a nucleic acid according to the invention is defined as a nucleic acid that differs from the nucleic acid of reference by one or a plurality of substitutions, additions or deletions of a nucleotide relative to the nucleic acid of reference. A variant of a nucleic acid according to the invention can be of natural origin such as an allelic variant that exists naturally. Such a nucleic acid variant can also be a non-natural nucleic acid obtained, for example, by mutagenetic methods.
In general, the differences between the nucleic acid of reference and the variant nucleic acid are reduced in such a way that the nucleic acid of reference and the variant nucleic acid have very similar nucleotide sequences and in a number of identical regions.
The regulator nucleic acid variants according to the invention comprise all 100 % nucleotide identity with the region running from nucleotide 1379 to nucleotide 1390 of sequence SEQ ID № 1. In addition, the trinucleotide of the variants of the regulatory nucleic acid according to the invention corresponds to the trinucleotide running from nucleotide 1486 to nucleotide 1488 of sequence SEQ ID № 1 codes always for an amino acid chosen form the group comprising alanine, isoleucine, and leucine.
Thus in the regulator nucleic acid according to the invention the trinucleotide running from nucleotide 1488 to nucleotide 1488 of sequence SEQ ID № 1 is chosen among the following trinucleotides :

(i) the following trinucleotides coding for the amino acid alanine : GCU,
GCT, GCC, GCA et GCG ; (i) the following trinucleotides coding for the amino acid isoleucine : AUU,
ATT, AUC, ATC, AUA and ATA ; and (iii) the following trinucleotides coding for the amino acid leucine : CUU,
CTT, CUC, CTC, CUA, CTA, CUG, et CTG.
A variant of a regulatory nucleic acid according to the invention conserves the capacity of directing the expression of a polynucleotide of specific interest in the cells of the posterior sericigenic glands of a lepidopteran. In order to verify the functional character of a "variant" of a nucleic acid according to the invention, including a nucleic acid comprising 90 % nucleotide identity with the sequence SEQ ID № 1, the man skilled in the art can implement the specific expression tests of a reporter or marker gene described in the examples.
A "polynucleotide of interest" in the meaning of the invention is defined as a polynucleotide capable of directing the synthesis of a polypeptide fusion between a modified signal peptide and a polypeptide of interest.
A nucleic acid according to the invention comprises a sequence with a promoter function, said promoter constituting a regulation signal directing the expression of a polynucleotide of interest, specifically in the cells of the posterior sericigenic gland of a lepidopteran.
For the purposes of the present description a nucleic acid having a "promoter" function, also called a "promoter" or.even "promoter sequence" consists of a nucleic acid that has the RNA polymerase recognition motifs and more specifically a "TATA" box and a "CAAT" box, whose structure is well-known to the specialist in the art.
It has been shown according to the invention that the nucleic acid sequence SEQ ID N° 1 comprises the regulation sequences necessary to the expression of a polynucleotide of interest, specifically in the cells of the posterior sericigenic glands of Bombyx mori. Such specificity was not observed with the regulatory sequences described by Tamura (Tamura et al. Germline transformation of the silkworm Bombyx mori L. using a piggyBac transposon derived vector. Nature Biotechnology Vol. 18, 2000) whose expression was not limited to the cells of the posterior sericigenic glands of Bombyx mori. It has also been shown according to the invention that the nucleic acid comprising the 5' terminus to the 3' terminus, the polynucleotide running from nucleotide 1150 to nucleotide 2026 of sequence SEQ ID № 1 is capable of directing the expression of a polynucleotide of interest specifically in the cells of the posterior sericigenic glands of Bombyx mori.
The sequence SEQ ID № 1 comprises, from the 5' terminus to the 3' terminus, the promoter of the fibrohexamerin gene between nucleotide 1 and nucleotide 1451.

This promoter comprises a "TATA" box located from the nucleotide in position 1420 the nucleotide in position 1423 of the sequence SEQ ID N° 1. This promoter sequence also has a "CAAT" box located from nucleotide at position 1252 up to the nucleotide at position 1365 of the sequence SEQ ID № 1. This promoter sequence then contains a sequence « TATTTATTTAA » located from the nucleotide in position 1379 to the nucleotide in position 1390 of the sequence SEQ ID № 1. The TATA box constitutes the promoter element itself, which is located in general at a distance of approximately 30 bases from the site of transcription initiation. The CAAT box is an element acting in cis that is found commonly in the promoter and activator region ("enhancer"). The sequence SEQ ID № 1 comprises the exon 1 of fibrohexamerin between the nucleotide in position 1452 and the nucleotide in position 1525, which codes for the modified signal peptide.
The sequence SEQ ID № 1 comprises the intron 1 of fibrohexamerin between the nucleotide in position 1526 and the nucleotide in position 2026. The sequence SEQ ID N° 1 comprises a polynucleotide between the nucleotide in position 2027 and the nucleotide in position 2040. This latter polynucleotide is not essential to the invention and has merely facilitated the construction of the sequence SEQ ID № 1 that comprises in addition a polynucleotide coding for a reporter protein "Ds-Red" between the nucleotide in position 2041 and the nucleotide in position 2721.
A nucleic acid comprising (i) the regulatory region running from the nucleotide in position 1 up to the nucleotide in position 1451 of sequence SEQ ID N° 1 or any biologically active fragment of this nucleic acid containing the TATA, CAAT boxes and at least one «CTATTTATTTAA » sequence as defined hereinbefore and (ii) a region coding for a modified signal peptide placed under the control of such a nucleic acid constitutes another object of the present invention.
It has been shown according to the invention that exon 1 of fibrohexamerin as defined hereinbefore should have a modification of the trinucleotide in position 1486 to 1488 of the sequence SEQ ID № 1, so that the addressing of the polypeptide coded by the polynucleotide of interest is correct; and so that the polypeptide coded by the polynucleotide of interest is secreted with the silk thread. The trinucleotide in position 1486 to 1488 of the sequence SEQ ID № 1 coding for cysteine must be modified into a trinucleotide coding for an amino acid chosen from the group comprising alanine, isoleucine and leucine. In virtue of the realization of constructions of DNA comprising the totality or the most restrictive parts of the sequence SEQ ID № 1, it has been shown according to the invention that the nucleic acid of the invention makes it possible to specifically direct the expression of a polynucleotide of interest in the cells of the posterior sericigenic glands ofBombyx mori. The presence of the Ds-Red reporter gene has also made it possible to show that the modification of the exon 1 of fibrohexamerin

makes it possible to obtain correct secretion of the polypeptide coded for by the volynucleotide of interest.
Expression cassettes comprising a polynucleotide of interest coding for a reporter gene, the gene of the Ds-Red protein, which has been placed under the control of a regulator nucleic acid as defined hereinbefore, comprising the specific tissue regulatory signals, exon 1 of modified fibrohexamerin, the entirety of intron 1 of fibrohexamerin and a nucleic acid with a promoter function.
These expression cassettes have been used for transforming cells of Bombyx mori and, more precisely the gamete precursors using a transgenic animal, after which the expression of the reporter gene has been followed in different tissues of the animal. The results show that the regulatory signals necessary to the activity of specific tissue transcription and strong expression in the cells of the posterior sericigenic glands of Bombyx mori are included especially in the region running from nucleotide 1150 to nucleotide 2026 of the sequence SEQ ID № 1.
More precisely, it has been shown according to the invention that this region contains a motif: « CTATTTATTTAA » (sequence SEQ ID№ 2) located from the nucleotide in position 1379 to the nucleotide in position 1390 of the sequence SEQ ID N° 1. This motif constitutes a binding site for SGF1 forkhead type transcription factors (Julien, E-, Bordeaux, M.C., Garel, A. & Couble, P. (2002 Fork head alternative binding drives stage specific expression in the silk gland of Bombyx mori. Insect Biochem Molecul. Biol. 32, 377 387.)
Without willing to be bound by any theory, the inventors believe that the motif «CTATTTATTTAA» constitutes an important structural characteristic of the nucleic acid of the invention, having a transcription sequence activator function ("enhancer") and a specific tissue expression regulator function.
According to the invention, novel motifs "CTATTTATTTAA" upstream of the motif naturally contained in the promoter of fibrohexamerin makes it possible to increase the rate of expression of the polypeptide coded by the polynucleotide of interest, without altering specificity.
The introduction of DNA carried by the piggyBac integration vector into the genome of Bombyx mori obtained after microinjection into the eggs of the animal has also made it possible to show in an unexpected fashion that the modification of the exon 1 of the fibrohexamerin gene makes it possible to obtain addressing of the protein of interest compatible with a secretion of same in the silk filament. In other words, when a polynucleotide of interest is fused in phase with the 3' terminus of the exon 1 of the fibrohexamerin gene, the modification of the exon 1 of this gene, which codes for the modified signal peptide, makes it possible to conserve secretion of the protein of interest identical to that of fibrohexamerin.

According to the invention, a motif of sequence SEQ ID № 10 or SEQ ID N° 11 •in be integrated into the promoter of fibrohexamerin so as to dimerize the Other expression activator sequences, homologous or heterologous, can be integrated in the fibrohexamerin promoter. By way of example, one can cite the sequences corresponding to the Gal4/UAS system of yeast, known to the man skilled in the art, and which will not be described in detail. One can also mention the sequences corresponding to the amplifier of the silk worm cytoplasmic actin gene (Mange A. Julien E. Prudhomme JC. and Couble P. A strong inhibitory element down-regulates BRE-stimulated transcription of the A3 cytoplasmic actin genes fBombyx mori. J Mol Biol. 1997 Jan 24;265(3):266-74. Fatyol K., Illes K. and Szalay A.A. An alternative intronic promoter of the Bombyx A3 cytoplasmic actin gene exhibits a high level of transcriptional activity in mammalian cells; Mol Gen Genet. 1999 Mar. 261 (2): 337-45). Finally, one can mention the sequences corresponding to the amplifier system of the baculovirus IEl/Hr5 or Hr3 (Lu M., Farrell P.J., Johnson R. and latrou K. J Biol Chem. 1997 Dec 5; 272 (49): 30724-8). Regulatory nucleic acids according to the invention.
The object of the invention relates to a nucleic acid directing the expression of a protein of interest specifically in the cells of the posterior sericigenic glands ofBombyx mori, wherein said nucleic acid comprises from the 5' terminus to the 3' terminus, (i) a regulatory region comprising regulatory signals for the expression of a polynucleotide of interest specifically in said sericigenic cells and (ii) a region coding for a modified signal peptide placed under the control of said regulatory region, said nucleic acid having at least 90 % identity in nucleotides with the polynucleotide running from nucleotide 1150 to nucleotide 2026 of sequence SEQ ID№ 1 and in which the trinucleotide running from the nucleotide 1486 to nucleotide 1488 of sequence SEQ ID NO 1 codes for an amino acid chosen from the group comprising alanine, isoleucine and leucine.
According to a first embodiment, particular to the aforementioned nucleic acid, said nucleic acid is characterized in that it comprises, from the 5' terminus to the 3' terminus, (i) a regulatory region comprising regulatory signals for the expression of a polynucleotide of interest specifically in said sericigenic cells and (ii) a region coding for a modified signal peptide, placed under the control of said regulatory region, said nucleic acid comprising, from the 5' terminus to the 3' terminus, the polynucleotide running from nucleotide 1150 to nucleotide 2026 of sequence SEQ ID № 1 and in

which the trinucleotide running form the nucleotide 1486 to the nucleotide 1488 of the Oquence SEQ ID NO 1 codes for an amino acid chosen from the group comprising alanine, isoleucine, and leucine.
According to a second embodiment, of the aforementioned nucleic acid, said nucleic acid is characterized in that it comprises, from the 5' terminus to the 3' terminus, (i) a regulatory region comprising regulatory signals for the expression of a polynucleotide of interest specifically in said sericigenic cells and (ii) a region coding for a modified signal peptide, placed under the control of said regulatory region, said nucleic acid comprising, from the 5' terminus to the 3' terminus, the polynucleotide running from nucleotide 1 to nucleotide 2026 of sequence SEQ ID № 1 and in which the trinucleotide running form the nucleotide 1486 to the nucleotide 1488 of sequence SEQ ID NO 1 codes for an amino acid chosen from the group comprising alanine, isoleucine, and leucine.
According to a third particular embodiment, the nucleic acid according to the invention consists of a nucleic acid such as described hereinbefore that has been modified by the introduction of at least one and not more than four copies of a polynucleotide of sequence SEQ ID №. 2 between the nucleotides 1 and 1379 of the sequence SEQ ID Na 1. For introducing a copy of a polynucleotide of sequence SEQ ID NO 2, one can insert between nucleotides 1 and 1379 and especially between nucleotides 1378 and 1379, of sequence SEQ ID № 1, a sequence SEQ Id № 10 or SEQ ID NO 11. The present invention also relates to a nucleic acid, whose sequence is complementary to the sequence of any one of the nucleic acids as defined hereinbefore. As has already been stated, one of the objectives pursued by the present invention is obtaining a high rate expression of a polypeptide in the cells of the posterior sericigenic cells ofBombyx mori coded by a polynucleotide of interest.
Further still, the object of the invention is a nucleic acid comprising a polynucleotide of interest coding for a polypeptide of interest, fused with the regulator nucleic acid directing the expression of the polypeptide of interest specifically in the posterior sericigenic cells ofBombyx mori as defined in the present description. Such a nucleic acid comprising a polynucleotide coding for a polypeptide of interest is also designated an "expression cassette" for the purposes of the present invention.
Further still, an object of the invention is an expression cassette comprising a polynucleotide of interest placed under the control of a regulator nucleic acid as defined hereinbefore. Expression Cassettes according to the invention
According to a first embodiment, an expression cassette according to the invention is characterized in that the polynucleotide of interest that it comprises codes for a polypeptide.

Alternatively, the polynucleotide of interest can code for a fusion polypeptide jmprising, from the N-terminal end to the C-terminal end :
- at least the 172 N-terminal amino acids of the fibroin L polypeptide
sequence (Tanaka K., Kajiyama N., Ishikura K., Waga 5., I ikuchi A,
Ohtomo K., Takagi T. and Mizuno S, Determination of the site of disulfide
linkage between heavy and light chains of silk fibroin produced by Bombyx
mori. Biochimica et Biophysica Anta 1432 (1999) 92-103), and
- a polypeptide of interest.
Preferably, the polypeptide of interest is chosen among the proteins such as spider spidroin (genus Nephila), Galleria fibroin or more generally any other polypeptide enabling the obtention of a silk filament, whose ductility strength or elasticity properties, for example, are modified.
The polynucleotide of interest can also code for a soluble polypeptide of biomedical interest such as, for example, the hormones, antigens, enzymes, growth factors, or even receptors.
In particular, the polynucleotide of interest can code for human interleukin 2.
An expression cassette according to the invention can be surrounded by insulators at one or at each end, intended to confer to the polynucleotide of interest an expression regardless of its site of integration. By way of example of insulators, one can mention the gypsy insulator of Drosophila (Gerasimova T.I. and Corces V. G.: Polycomb and trithorax group proteins mediate the function of a chromatin Insulator. Cell 1998, 92: 511-521), scs/scs1 (Kellum R. and Schedl P. 1991 A position effect assay for boundaries of higher order chromosomal domains. Cell 64: 941-950) or even the 5'H S4 insulator of the chicken beta globin gene (Chung J.H., Bell A. c. and Felsenfeld G. (1997) Characterization of the chicken beta-globin insulator. Proc. Nat. Acad. Sci, USA 94: 575-580). Recombinant vectors according to the invention
The invention relates to recombinant integration vectors comprising a regulator nucleic acid or an expression cassette as defined hereinbefore. The invention thus consists in recombinant vectors, in which a regulator nucleic acid according to the invention has been inserted and comprising regulation signals making possible the expression of a polynucleotide of interest, specifically in the cells of the posterior sericigenic glands of Bombyx mori, when this polynucleotide is placed under its control.
Also parts of the invention are recombinant integration vectors comprising
a) a regulator nucleic acid as defined in the present specification, and
b) a polynucleotide of interest placed under the control of the regulator nucleic
acid defined in a).

The polynucleotide of interest can consist in a polynucleotide coding for a stectable polypeptide or a marker polypeptide such as, for example, a polypeptide coding for the Ds-Red protein.
A recombinant integration vector according to the invention responding to the above definition is the Pig fhx(sp*)DsRed-(3xP3-GFP) deposited on February 20, 2003 in the Collection Nationale de Cultures de Micro-organismes (CNCM) of the Institut Pasteur under registration number CNCM 1-2975.
i
The invention also relates to recombinant cloning vectors comprising the nucleotide sequence of a recombinant integration vector such as that described above.
For obtaining any one of the regulatory sequences according to the invention, it is possible to utilize the techniques and primers described in the examples. The specialist in the art can also utilize the Pig fhx(sp*)DsRed-(3xP3- FP) deposited on February 20, 2003 in the National Collection of Microorganism Cultures (CNCM) of the Institute Pasteur under registration № CNCM 1-2975. General characteristics of the recombinant vectors according to the invention
"Vector" in the meaning of the present invention is defined as a molecule of circular DNA or RNA which is optionally in a single - stranded or double - stranded form. A vector according to the invention can be a cloning vector or an integration vector.
According to one preferred embodiment of the invention, a recombinant vector according to the invention is an integrative vector enabling insertion of copies of a DNA sequence in this vector into the genome of the animal of the Bombyx mori species.
Advantageously, the recombinant vector or, in other cases, the expression cassette contained in the vector can also contain the untranslated 3' sequences known as "terminators". Among the terminators that can be used in the constructions of the invention, the SV40 polyadenylation sequence can be mentioned. The preferred integration vector according to the invention is derived from the piggyBac transposon obtained from Trichoplusia ni, existing in the natural state in this species (Gary, L, C, et al. Transposon mutagenesis of baculoviruses: analysis of Trichoplusia ni transposon IFP2 insertions within the FF-locus of nuclear polyhedrosis viruses, Virology 172, 156-69(1989))
The piggyBac transposon comprises fragments of DNA able to move from one locus of the genome to another part on the basis of transposition. It comprises right and left "feet", comprised of two short reverse repeat sequences framing the genes coding for its mobility functions.
The integrative vector described according to the invention is a modified piggyBac transposon in which the nucleic acid of interest replaces a part of the genes

coding for the mobility functions of the transposon that have been removed.
The integrative vector is used in combination with an helper vector enabling the expression of the mobility genes suppressed in the integration vector. The integration vector combined with the helper vector makes possible the integration of the nucleic acid or the expression box according to the invention into the genome of the host cell on the basis of transposition.
The integrative vector according to the invention can also be derived from the Hobo element of Drosophila or from the minos transposon ofDrosophila.
According to another embodiment, a recombinant cloning vector is used in order to amplify the nucleotide sequence of an integrative vector as described hereinbefore. According to this other embodiment, the aforementioned recombinant integrative vector is linearized prior to its insertion into the receiver vector at a cloning site or a polysite of said receiver vector before ligation. The cloning vectors used are of bacterial or viral origin. They are, for example, PUC, pBR or pSK type plasmids known to the specialist in the art but will not be described in detail. Recombinant host cells according to the invention
In order to enable the expression of a polynucleotide of interest placed under the control of a regulator nucleic acid according to the invention, the regulator nucleic acids, the expression cassettes or even the recombinant vectors defined in the present description must be introduced into the host cell. The introduction of the polynucleotides according to the invention into a host cell can be done in vitro using methods well-known to the specialist in the art. "Host cell comprising a nucleic acid" is defined as a cell having integrated into its genome one or a plurality of copies of a nucleic acid.
In addition, a further object of the invention is a host cell comprising a regulator nucleic acid, an expression cassette or even a recombinant vector as defined hereinbefore. This host cell is preferably an animal cell belonging to the genus Bombyx, more particular to the species Bombyx mori, regardless of the strain considered. The host cell can also be a cell of an animal belonging to another lepidopteran species such as Philosomia cynthia or more generally to the genus Bombyx, Antheraea, Galleria, Philosamia, Spodoptera or Drosophila.
More preferably, a host cell according to the invention consists in a cell of the posterior sericigenic gland of Bombyx mori. Transgenic animals according to the invention
Further still, the object of the invention is the use of a nucleic acid, an expression cassette, a vector or a recombinant host cell as defined hereinbefore for obtaining a transgenic non-human animal of the genus Bombyx producing silk.
Preferably, said transgenic animal belongs to the Bombyx mori species and

secretes a protein of interest in the silk filament. The invention also relates to a ™oilticellular transgenic organism comprising a host cell or a plurality of host cells, an expression cassette or even a recombinant vector as defined hereinbefore. A further object of the invention is a transgenic non-human animal, comprising in an integrated form in its genome, a nucleic acid or an expression cassette as defined in the present description.
Generally, the invention relates also to the use of a regulator nucleic acid, an expression cassette, a recombinant vector or even a host cell as defined hereinbefore for obtaining a transgenic animal.
The invention relates also to a transgenic non-human animal, deficient for the secretion of fibroin L, comprising in a form integrated into its genome an expression cassette comprising a polynucleotide of interest coding for a fusion polypeptide comprising form the N-terminal end to the C-terminal end
- at least the 172 N - Terminal amino acids of the fibroin L polypeptide
sequence, and
- a polypeptide of interest.
Silk worms deficient in fibroin L are known. By way of example one can mention the Nd-s or Nd-sD mutants (Kazuyuki Mori, Kazunori Tanaka, Yoshimi Kikuchi, Miho Waga, Shou Wage and Shigeki Mizuno : production of a chimeric fibroin light-chain polypeptide in a fibroin secretion - deficient naked pupa mutant of the silkworm Bombyx mori. J. Mol. Biol. (1995) 251, 217-228.)
Nonetheless, the man skilled in the art can have recourse to classical techniques of genetic engineering for providing an animal deficient in fibroin L.
By way of example, the man skilled in the art wanting to realize an animal deficient in fibroin L can produce mutagenesis in silk worms, followed by a screening of the animals deficient in fibroin L. The mutagenesis can consist of a classical mutagenesis using UV or chemical mutagenesis or even directed mutagenesis of the fibroin L gene.
The screening of the animals deficient in fibroin L can be achieved by carrying out a Northern blot or a Western blot on the cocoons.
Alternatively, the man skilled in the art wanting to produce an animal deficient in fibroin L may also have access to the RNA interference technique. Generally, the fibroin produced by the posterior cells of the sericigenic gland (secretor) is a complex of three different polypeptides : a heavy chain (fibroin H), a light chain (fibroin L) and fibrohexamerin. The linkage by a disulfide bridge of fibroin H and fibroin L is essential to secretion of fibroin into the lumen of the gland. Fibroin L produced by the mutant homozygotes Nd-s or Nd-sD has a C-terminal region different from that produced by the wild type and this mutated fibroin L cannot form the disulfide bridge

b);
d) selection of the homozygote animals for the transgene. The invention has the further object of a method for obtaining a silk fiber, characterized in that it comprises the following steps :
a) breeding a transgenic animal as defined in the present specification;
b) recovering the silk produced by the transgenic animal.
The invention has the further object of a method for obtaining a protein of interest, characterized in that it comprises the following steps :
a) breeding a transgenic animal as defined in the present specification;
b) recovering the silk produced by the transgenic animal
c) recovering the protein of interest from the silk fiber.
Supplemental nucleic acids according to the invention
The invention also relates to a nucleic acid directing the expression of an RNA of interest reducing the synthesis of fibroin H, specifically in the cells of the posterior sericigenic glands of Bombyx mori, wherein said nucleic acid comprises a regulatory region comprising regulatory signals for the expression of a polynucleotide of interest specifically in said sericigenic cells, said nucleic acid having at least 90 % nucleotide identity with the polynucleotide running from nucleotide 1150 to nucleotide 1451 of the sequence SEQ ID .№ 1.
This type of nucleic acid responds to the set of general definitions that have been given in the first part of the description and, in particular, to the definition of percent identity between two sequences.
The success of the production of an exogenous protein by the cells of the sericigenic glands is associated inter alia with proteosynthesis that this protein will mobilize for its synthesis.
One way of optimizing the rate of expression of the alien protein can consist of diminishing expression of one of the silk proteins. Among these, fibroin H alone represents 80 % of the proteins produced by the sericigenic gland and is the major protein of silk. Because of its structure, it is responsible for physio-chemical and mechanical properties of the silk fiber : strength, elasticity, insolubility.
Elimination of this protein and its replacement by a protein of interest such as spidroin, fibroin H Galleria or other proteins of interest already mentioned enables especially from the modification of the properties of the silk fiber and thus the creation of new biomaterials.
In order to stably inhibit (thus transmissible to the descendence) the fibroin H gene in the posterior part of the sericigenic gland, an approach using RNA interference (iRNA), has been preferred. This apparently universal phenomenon since proved both in prokaryotes and in the eukaryotes consists in a specific and efficacious inhibition of

with fibroin H (due to the fact of the elimination by intronic recombination of the exon that contains the cysteine involved in the disulfide link). This secretion deficiency does not alter the viability of the animals but leads in the mutant silk worm to a delay of development of the secretor and production of a very fine cocoon (less than 0.3 % of fibroin compared to a normal cocoon) made essentially of sericins.
Thus, the invention relates to a transgenic non-human animal, deficient for the secretion of fibroin L, comprising in integrated form in its genome an expression cassette comprising a polynucleotide of interest coding for a fusion polypeptide comprising, from the N-terminal end to the C-terminal end :
- at least the 172 N-terminal amino acids of the fibroin L polypeptide
sequence, and
- a polypeptide of interest.
This particular embodiment of the invention makes available a transgenic animal in which the quantity of protein of interest produced is controlled in virtue of the presence of part of the fibroin L in the fusion polypeptide and of the absence of fibroin L naturally produced by the animal.
The transgenic animals according to the invention belong preferably to a species and genus that has already been listed above concerning the origin of the animal host cells of the invention. Furthermore, the object of the invention is a cocoon comprising a protein of interest, whose expression is directed by a nucleic acid, an expression cassette or a vector as described above and, wherein said protein of interest is present at a concentration in excess of 5 ng per mg of cocoon and preferably greater than 70 ng permg of cocoon.
A cocoon of 141 mg comprising between 1 and 10 μg of human interleukin-2 has been produced by a transgenic animal according to the invention in accordance with the protocol described in Example 7.
A further object is a method for obtaining a transgenic animal of the Bombyx mori species, characterized in that it comprises the following steps :
a) injection of a nucleic acid, an expression cassette or a vector as described
hereinbefore into the eggs of Bombyx mori between one and five hours
after the eggs are laid, prior to the formation of the blastoderm;
b) selection of the transgenic animals having integrated into their genome a
nucleic acid or an expression cassette as defined in the present
description.
. A further object of the invention is a method for obtaining a transgenic animal of the species Bombyx mori, characterized in that it comprises in addition the following steps :
c) crossing among themselves of two transgenic animals as obtained in step

a gene by degradation of its mRNA in response to exogenous double - stranded RNA.
The RNA of interest used can be an RNA of the siRNA type (small interference RNA) capable of pairing with mRNA from fibroin H in order to form a double -stranded RNA or a shRNA (small hairpin RNA) able to form a double - stranded RNA without pairing with the messenger RNA of fibroin H.
Preferably, the nucleotide sequence coding for the RNA of interest is the sequence SEQ ID №12.
The sequence SEQ ID № 12 is composed of an inversed and repeat nucleotide sequence derived from the fibroin H gene and will be discussed in more detail in Example 9.
The inventors have found that the transformation of Bombyx mori using a nucleic acid as described hereinbefore enables a significant reduction of the production of fibroin H as shown in Example 9 and Figure 13. This reduction of protein synthesis is based on the principle of interference RNA, a post-transcriptional process triggered by the introduction of double - stranded RNA in a cell, leading to the inactivation or "invalidation" of a gene in a sequence - specific manner.
Recourse to such a nucleic acid enables specifically the reduction of the production of fibroin H, without affecting the production of other proteins entering into the composition of the silk fiber.
Without intending to be bound by a particular theory, the inventors believe that the RNA of interest forms a hairpin structure, able to destabilize the RNA polymerase and thus reducing the synthesis of fibroin H.
The reduction of the rate of Bombyx mori synthesis of fibroin H has stimulated considerable interest in the context of the present invention. Recourse to the nucleic acids described immediately hereinabove, in combination with those directing the expression of a polypeptide of interest, provides animals in which the cellular resources are redirected towards the production of the protein of interest. This embodiment of the invention responds fully to the need for the availability of an industrial tool for the production of recombinant proteins -in large quantities. Transgenic animals comprise several types of nucleic acids such as those described hereinbefore and which will be described in more detail in the following description. Preferably, the nucleic acid according to the invention comprises a regulatory region comprising regulatory signals for the expression of a polynucleptide of interest \ specifically in said sericigenic cells, said nucleic acid comprising, frorn the 5' terminus to the 3' terminus; the polynucleotide running from nucleotide 1150 to nucleotide 1451 of the sequence SEQ ID № 1.
Preferably, the nucleic acid according to the invention comprises a regulatory region comprising regulatory signals for the expression of a polynucleotide of interest

specifically in said sericigenic cells, said nucleic acid comprising, from 5' terminus to e 3' terminus; the polynucleotide running from nucleotide 1 to nucleotide 1451 of the sequence SEQ ID № 1.
A further object of the invention is a nucleic acid consisting of a nucleic acid such as that defined hereinbefore that has been modified by the introduction of at leas one and not more than four copies of a polynucleotide having the sequence SEQ ID N» 2,between nucleotides 1 and 1379 of the sequence SEQ ID№ 1.
The invention also relates to a nucleic acid having a sequence complementary to a nucleic acid such as that described hereinbefore. Supplemental expression cassette according to the invention
The invention also relates to an expression box comprising a nucleic acid coding for an RNA of interest and placed under the control of a nucleic acid such as described hereinbefore.
Preferably the RNA of interest is coded by a nucleic acid having the sequence SEQ ID KO 12.
An expression box according to the invention can be surrounded by insulators at one or at each end, intended to confer to the polynucleotide of interest expression regardless of its site of integration. By way of example of insulators, one can mention the gypsy insulator of Drosophila (Gerasimova T.I. and Corces V.G. : Polycomb and trithorax group proteins mediate the function of a chromatin insulator, Cell 1998, 92: 511-521), scs/scs1 (Kellum R. and Schedl P. 1991 A position effect assay for boundaries of higher order chromosomal domains. Cell 64: 941-960) or even the 5'H S4 insulator of the chicken beta globin gene (Chung J.H., Bell A. c. and Felsenfeld G. (1997) Characterisation of the chicken beta-globin insulator. Proc. Nat. Acad. Sci, USA 94: 575-580). Supplementary vectors according to the invention
Another object of the invention is a recombinant integrative vector comprising a nucleic acid or an expression cassette such as that described hereinbefore.
The invention thus consists in recombinant vectors, in which a regulator nucleic acid according to the invention has been inserted and comprising regulation signals enabling the expression of a RNA of interest reducing the synthesis of fibroin H, specifically in the cells of the posterior sericigenic glands of Bombyx mori, when this polynucleotide is placed under its control. Also forming part of the invention are recombinant integrative vectors comprising :
a) a regulator nucleic acid as defined hereinbefore, and
b) a nucleic acid coding for an RNA of interest placed under the control of
the regulator nucleic acid defined in a).
A recombinant integrative vector according to the invention responds to the

general definitions of vectors that have been described in the section entitled "General characteristics of recombinant vectors according to the invention."
The invention also relates to recombinant cloning vectors comprising the nucleotide sequence of a recombinant integration vector such as that described above.
For obtaining any one of the regulatory sequences according to the invention, it is possible to utilize the techniques and primers described in the examples. The specialist in the art, replacing only the nucleic acid sequence directing the synthesis of the DS-red protein with a sequence directing the synthesis of an RNA of interest reducing the synthesis of fibroin H. Recombinant host cells according to the invention
In order to enable the expression of an RNA reducing the synthesis of fibroin H placed under the control of a regulator nucleic acid as described above, the regulator nucleic acids, the expression cassettes or even the recombinant vectors defined hereinbefore must be introduced into the host cell.
The introduction of the polynucleotides according to the invention into a host cell can be done in vitro using methods well-known by the man skilled in the art. "Host cell comprising a nucleic acid" is defined as a cell having integrated into its genome one or a plurality of copies of a nucleic acid.
In addition, a further object of the invention is a host cell comprising a regulator nucleic acid, an expression cassette or even a recombinant vector as defined hereinbefore.
This host cell is preferably an animal cell belonging to the genus Bombyx, more particular to the species Bombyx mori, regardless of the strain considered. The host cell can also be a cell of an animal belonging to another lepidopteran species such as Philosomia cynthia or more generally to the genus Bombyx, Antheraea, Galleria, Philosamia, Spodoptera or Drosophila.
More preferably, a host cell according to the invention consists in a cell of the posterior sericigenic gland of Bombyx mori. Supplementary transgenic animals according to the invention
Further still, the object of the invention is the use of a nucleic acid, an expression cassette, a vector as defined hereinbefore for obtaining a transgenic animal of the genus Bombyx.
Preferably, said transgenic animal belongs to the Bombyx mori species and secretes a protein of interest in the silk filament.
The invention also relates to a multicellular non-human transgenic organism comprising a host cell or a plurality of host cells, an expression cassette or even a recombinant vector as defined hereinbefore.
Further still, the object of the invention is a non-human transgenic animal

comprising, in a form integrated into its genome :
(i.) a nucleic acid, an expression cassette or a vector such as described in the first part of the description, directing the expression and the secretion of a protein of interest and
(ii.) a nucleic acid, an expression cassette or a vector as defined in the second part of the description, directing the synthesis of an RNA of interest reducing the synthesis of fibroin H.
The availability of a transgenic animal as described above is of considerable industrial interest. In fact, in such an animal, the reduction of the synthesis of fibroin H enables the redirection of cellular resources to the synthesis of the protein of interest without inasmuch affecting the secretion of the silk fiber. This embodiment of the invention responds fully to the need for the availability of an industrial tool for the production of recombinant proteins in large quantities.
The invention also relates to a non-human transgenic animal: (i.) deficient for the secretion of fibroin L,
(ii.) comprising in a form integrated into its genome an expression cassette, directing the expression of a fusion protein between at least the 172 N-terminus amino acids of the polypeptide sequence of fibroin L and a peptide of interest, and
(iii.) a nucleic acid, an expression cassette or a vector as defined in the second part of the description, directing the synthesis of an RNA of interest, reducing the synthesis of fibroin H.
This particular embodiment of the invention provides a transgenic animal having the set of advantages associated with the invention; in other words, an animal, wherein the quantity of the protein of interest produced is controlled and wherein the cellular resources are oriented towards the synthesis of a protein of interest. Accordingly, the invention provides a directly utilizable industrial tool for the production of recombinant proteins on a large scale at reduced cost.
Generally, the invention relates also to the use of a regulator nucleic acid, an expression cassette, a recombinant vector or even a host cell as defined hereinbefore for obtaining a non-human transgenic animal.
The transgenic animals described hereinbefore belong preferably to a species and genus that has already been listed above, concerning the origin of the animal host cells of the invention.
The transgenic animals described hereinbefore can be obtained according to methods identical to those which have been described in the first part of the description.
In order to be able to easily distinguish the animals comprising at least one

nucleic acid as described in the present application, the inventors have also developed aroup of primers described in the following.
The sense primer, comprises the 5' terminus three nucleotides corresponding to the nucleotides coding for alanine, isoleucine or leucine.
The antisense primer is chosen by any means, since it hybridizes on the complementary strand, at 3' to the sense primer.
The recourse to primers during polymerase chain reaction (PCR), done under stringently controlled conditions enables only the amplification of DNA sequences comprising a mutation of the amino acid cysteine into alanine, isoleucine or leucine.
Accordingly, the transgenic animals can be characterized by the presence of a band in Southern blot that is not present for the wild type animals.
One can also use the sequences SEQ ID № 13 and SEQ ID № 14 for amplifying a nucleotide sequence, then detecting the presence or absence of a mutated cysteine residue in the product of said amplification by using a probe such as defined hereinbefore.
In addition, the invention is illustrated but by no means limited by the figures and the following examples, wherein :
Figure 1 represents a protocol for constructing a vector according to the
invention;
Figure 2 represents a protocol for substituting the cysteine codon with an
isoleucine codon in the signal peptide of the gene coding for firbrohexamerin;
Figure 3 represents a protocol for modifying the promoter of fibrohexamerin by
the addition of three copies of a polynucleotide of the sequence SEQ ID NO 2;
Figure 4 diagrammatically represents the sequence SEQ ID№ 3;
Figure 5 represents the specific tissue accumulation of the Ds-Red protein
placed under the control of a nucleic acid according to the invention;
Figure 6 represents the detection of the Ds-Red protein placed under the control
of a nucleic acid according to the invention in the sericigenic glands of a silk
worm;
Figure 7 represents the specific accumulation in the silk fiber of the Ds-Red
protein placed under the control of a nucleic acid according to the invention;
Figure 8 represents the detection of the Ds-Red protein placed under the control
of a nucleic acid according to the invention in the cocoons of the silk worm;
Figure 9 represents the presence of the Ds-Red protein at the surface of the fiber
on observation of the fiber using scanning electron microscopy; The protein
appears in the form of globular particles of variable size (depending on the
number of aggregates) coating the fiber.
Figure 10 represents the detection of the IL-2 protein placed under the control

of a nucleic acid according to the invention in the cocoons of the transgenic
heterozygous silk worm;
Figure 11 represents the detection of the fibroin H protein of Galleria
mellonella placed under the control of a nucleic acid according to the invention
in the sericigenic glands (A) and the cocoons (B) of heterozygous transgenic
silk worms;
Figure 12 represents a protocol for constructing a vector comprising a nucleic
acid directing the expression of an RNA reducing the synthesis of fibroin H;
Figure 13 represents the reduction of the synthesis of fibroin H of animals
transformed using the vector represented in Figure 12.
EXAMPLES General materials and methods generally used in the examples
A) Materials
The animals used belong to the species Bombyx mori, Nistari strain form the Unite Nationale Sericole (25 quai Jean-Jacques Rousseau, 69350, La Mulatiere, France). It is a multivoltine strain developing without embryonic diapause.
The bacteria used for clonings are E. coli DH5 alpha-Ti Max. Efficiency available from the INVITROGEN company (reference 440097).
B) Methods
* Breeding conditions of the animals
The silk worms were raised under controlled conditions in an "Animalerie Al" animal pen, at variable temperatures depending on the age of the larvae (between 22 °C and 25 °C), and at 75 % humidity. They were fed with artificial medium prepared on the basis of a granulate of mulberry leaves.
* Extraction of the nucleic acids
The DNA of the plasmid constructions was extracted from the bacteria using the Qiaprep Spin Miniprep from Qiagen (reference 27 106). Genomic DNA from the silk worms was extracted using phenol - chloroform - isoamyl alcohol. Silk worm genomic DNA was extracted using phenol chloroform isoamyl alcohol.
* Production of transgenic animals
Injection of the integrative vector into the eggs of the animal is done 2 to 5 hours after laying, prior to the formation of the blastoderm. A solution comprising the integrative vector and the "Helper" vector in equal quantities is prepared in a 0.5 mM phosphate buffer (pH 7.0) containing 5 mM KC1. Using an Eppendorf microinjector (Transjector 5246), 1 to 5 nL of this solution are injected with an Eppendorf microinjector (Transjector 5246). The hole formed by the injection is then covered using a glue (Loctite Superglue 3, Henkel France S.A.) and the eggs are kept at 25 °C in order that they continue their development.

* Selection of the transgenic animals
Selection of the transgenic animals with the Pigfhx(sp*)DsRed-(3xP3-GFP) Sector is done in virtue of the early expression of the 3xP3-GFP gene in the eyes of the animal.
* Detection test for the presence of the Ds-Red protein in the transgenic animal
The Ds-Red protein is visualized in situ (in the cells of the sericigenic gland) and in the cocoon under a Leica fluorescence magnifier (MZFL2) equipped with an excitation light (558 nm) emission system and an emitting light filter at 582 nm and a digital capture device. Analysis of cocoon fluorescence is done dry. The desiccated sericigenic glands are observed under the magnifier in some PBS. Detection of the Ds-Red protein was done at different stages of development and in particular at the 5th larval stage at the time of massive synthesis of silk proteins.
Detection was done by Western blot using a double antibody on the protein extracts from the sericigenic glands or from the silk cocoons : a rabbit anti-Ds-Red antibody (Clonetech, reference 8370-1) and a goat anti-rabbit antibody coupled to peroxidase (Biorad, reference 170-6515).
* Detection test for Ds-Red protein by Western blot realized with samples from the
sericigenic glands, cocoon and silk worm blaze.
The detection test for Ds-Red protein by Western blot comprises several steps:
- Extraction of the protein samples
A third of the cocoon or the pair of sericigenic glands (secretor separated from the reservoir) are taken and transferred into a microtube. The cocoon is then broken up into small pieces or the gland is crushed in 1 mL of LiSCN 10M; then the protein extracts are incubated for at least 1 hour at room temperature, while stirring periodically using a vortex. The protein samples are then diluted in 4/5 Tris buffer 10 mM - 2 % SDS - 5 % (3-mercaptoethanol.
The exterior envelope of the cocoon or blaze is transferred into a microtube to which 150 μL H2O are added for rinsing the blaze. The soluble Ds-Red protein is recovered by exposing the sample to ultrasounds for 10 minutes.
The samples are prepared prior deposit by denaturing the protein extracts in Laemmli 5x buffer for 5 minutes at 100 °C.
- Electrophoresis
The protein extracts are deposited on pre-formed gels (INVITROGEN Novex Tris glycine, 10 wells, 1.0 mm). Migration of the proteins is done at 120 V, 40 mA for 2h 30 min using a migration buffer (INVITROGEN Novex SDS Running buffer (10X)).
- Proteins transfer
The proteins are transferred to 9 cm by 6 cm nitrocellulose membranes (Hybond

ECL Amersham Pharmacia). The size of the whatman paper used is 10 cm by 7.5 cm. Transfer of the proteins is done at 120 V, 250 mA for Ih 15 min using a transfer buffer CHI Vitrogen Novex Tris Glycine Transfer Buffer (25X)).
- Staining of the proteins with Ponceau red
The nitrocellulose membrane is incubated with ponceau red (Sigma) for 1 minute in order to make the transferred proteins visible. If necessary, the size marker bands are marked with a pen.
- Blocking of the membrane
The membrane is incubated in PLT (PBS - 0.2 % Tween - 2 % milk) over night a4°C.
- Incubation with the first antibody
The first anti-Ds-Red antibody (Living Color DsPeptide Clontech) is diluted in PLT to 1/500 and it is incubated for 2 hours at room temperature. Incubation is followed by rinsing twice for 5 minutes in PBS - 0.02 % tween.
- Incubation with the second antibody
The secondary antibody (goat anti-rabbit IgG (H + L), horseradish peroxidase conjugate, Biorad) is diluted in PLT to 1/3000 and incubated for 1 hour at ambient temperature. Incubation is followed by rinsing 4 times for 10 minutes in PBS - 0.02 % tween.
- Development of the membranes
An A solution (5 mL of Tris 100 mM, pH 8.5, 22 μL coumaric acid and 50 jal of luminol added in the dark) and a solution B (5 mL of Tris 100 mM, pH 8.5, 3 fiL of H2O2). Extemporaneously, a mixture of solutions A and B is prepared in the dark, then the membrane is incubated to develop for 1 minute in the A and B solution. The membrane is exposed on an autoradiographic film for about 3 minutes and the film is developed after exposure.
Example 1 : Construction of the integrative vector comprising an expression cassette comprising a polynucleotide coding for the Ds-Red polypeptide placed under the control of a regulator nucleic acid according to the invention and, in particular, construction of the expression cassette comprising one part of the sequence SEQ ID N° 1.
The first step in the construction is represented in Figure 1A. Two plasmids are necessary for this first step.
It is a pDsRedl-Nl supplied by Clonetech (reference 6921-1), comprising from its 5' terminus to the 3' terminus :
- the CMV IE promoter;
- the Ds-Red gene;
- the SV40 polyadenylation signal;

- the kanamycin resistance gene;
- the replication origin of the pUC plasmid.
The Ds-Red protein gene is delimited at its ends by the nucleotide sequence of the Age 1 and Not 1 restriction enzymes.
The second plasmid used, called pfhx(sp*)GFP, comprises from its 5' terminus to the 3' terminus :
- the promoter of the modified fibrohexamerin;
- the gene coding for green fluorescent protein (GFP);
- the SV40 polyadenylation signal;
- the kanamycin resistance gene;
- the replication origin of the pUC plasmid.
As represented in Figure 1A, the pfhx(sp*)GFP plasmid comprises also a nucleotide sequence delimited by the nucleotide sequence of two restriction enzymes, Age 1 and Not 1, placed downstream of the promoter sequence of the fibrohexamerin gene. The Age 1 -Not 1 fragment obtained from the pDSredl-Nl and comprising the sequence coding for the Ds-Red Protein is cloned in the pfhx(sp*)GFP plasmid, in the position of the Agel -Not 1 sequence.
The plasmid resulting from this cloning, called pfhx(sp*)DsRed is represented in Figure IB and comprises, from the 5' terminus to the 3' terminus :
- the promoter of the modified fibrohexamerin;
- The Ds-Red gene;
- the SV40 polyadenylation signal;
- the kanamycin resistance gene;
- the replication origin of the pUC plasmid.
As represented in Figure IB, the promoter of the modified fibrohexamerin and the Ds-Red gene are included in a fragment delimited by the Bgl II and the Nael sequences. This fragment is cloned in the pPigA3cyp6a2-(3xP3-GFP)2 vector derived form the piggyBac transposon, whose structure is represented in Figure IB, in the position of the Bgl II - Stul fragment included in said latter, using a method identical to that described hereinbefore. The pPigA3cyp6a2(3xP3-GFP)2 vector comprises a left foot (L) and a right foot (R), represented in Figure IB.
The vector obtained at the end of this second cloning, represented in Figure 1C, called Pigfhx(sp*)DsRed-(3xP3-GFP) responds to the definition of a vector according to invention.
Example 2 : Method for substituting the cysteine codon with an isoleucine codon into the gene coding for the signal peptide of the fibrohexamerin. The method used is represented in the figure 2 and comprises several steps. First of all, a fragment of the fibrohexamerin promoter is isolated by digestion by the Hindlll and Pst I restriction

enzymes. The second step, designated PCR1 in Figure 2A, consists of performing a polymerase chain reaction using the primers of the sequence SEQ ID Ns 4 and SEQ ID № 5 represented in Figure 2A. The third step designated PCR2 in Figure 2A, consists of performing a polymerase chain reaction using the primers of the sequences SEQ ID NO 6 and SEQ ID № 7.The primers of the sequence SEQ ID № 5 and SEQ ID № 6 comprise an isoleucine codon for replacing the cysteine codon of the natural signal peptide of fibrohexamerin. The amplified nucleic acids are then ligated and the nucleic acid represented in Figure 2B is obtained, delimited by the sequences of the SacII and PstI restriction enzymes. This nucleic acid comprises an isoleucine codon represented in bolded type in Figure IB, replacing the natural cysteine codon. The fragment is then cloned instead of the corresponding natural sequence in the pfhx(sp*)GFP plasmid. Example 3 : Protocol for modifying the promoter of fibrohexamerin by the addition of three copies of a polynucleotide of sequence SEQ ID №2, binding the SGF-1/forkhead transcription factor, between nucleotides 1 and 1279 of the sequence SEQ ID № 1; this protocol is represented in Figure 3.
In the first step, a polymerase chain reaction of the fibrohexamerin promoter sequence is done. This amplification is realized using the A, B, C and D primers, represented in Figure 3, of the sequence SEQ ID № 3 for the B primer and the sequence SEQ ID N° 9 for the C primer.
In a second step, the nucleic acids obtained are subjected to ligation in order to obtain the nucleic acid, whose coding strand is represented in Figure 3.
The nucleic acid obtained comprises one part of the promoter sequence of the fibrohexamerin modified by the addition of three copies of a polynucleotide of sequence SEQ ID N° 2. This nucleic acid is then cloned into the pfhx(sp*)GFP plasmid in the position of the natural sequence of the fibrohexamerin promoter delimited by the Hind III and Pst I restriction sites.
The drawing of the sequence SEQ ID № 3 is represented in Figure 4.
The sequence SEQ ID N° 3 comprises, from the 5' terminus to the 3' terminus :the promoter (1) of the fibrohexamerin gene;
- three attachment sequences (2) of the SGFl/forkhead element (3) in addition to
the natural sequence;
- the fibrohexamerin exon 1 (4), whose cysteine codon is modified;
- the fibrohexamerin intron 1 (5);
- a polynucleotide (6) having helped the construction of the sequence SEQ ID № 1;
- a polynucleotide (7) coding for the Ds-Red reporter protein.
Example 4 : Ds-Red protein accumulation in the sericigenic glands of the silk worm rendered transgenic using the pPigFbx(sp*)DsRed-(3xP3-GFP) vector derived from the piggyBac transposon comprising the sequence SEQ ID № 1. The photographs a, b,

c, d, e and f represented in Figure 5, represent the tissue-specific expression of the Ds-Red protein in a silkworm rendered transgenic using the vector derived from the piggyBac transposon comprising the sequence SEQ ID № 3. Photographs a, b and c were made in daylight in three enlargements. The test for detecting the presence of the Ds-Red protein was done using the protocol described hereinbefore. Photographs d, e and f are the shots corresponding to photographs a, b and c but made in the fluorescent spectrum of the Ds-Red protein.
These photos allow to confirm that the fluorescence of the Ds-Red protein and thus its expression is limited to the posterior sericigenic cells (PSG, 2) of Bombyx mori.
No fluorescence corresponding to the Ds-Red protein presence was observed in the median sericigenic cells (SSG, 1). It can be deduced that the nucleic acids according to the invention drives the expression of the polypeptide of interest specifically in the posterior sericigenic cells of a lepidopteran.
This result is confirmed by the Western blot represented in Figure 6. This Western blot done under the conditions described hereinbefore helps the detection of the presence of the Ds-Red protein in the sericigenic cells of the silk worm. An SDS-PAGE gel (14 %; Tris - glycine; 0.1 % SDS) was transferred to a nitrocellulose filter and stained with Coomassie blue. The nitrocellulose filter is shown in Figure 6A. This same filter was then incubated with a primary polyclonal anti-Ds-Red antibody and then using a secondary goat anti-rabbit antibody conjugated with horseradish peroxidase. The result is shown in Figure 6B. Bands detected by chemoluminescence correspond to the presence of the Ds-Red protein.
Four deposits of proteins from the sericigenic glands of a silk worm can be seen on the filter shown in Figure 6A and 6B. The proteins were extracted from the sericigenic glands suing the protocol described hereinbefore. The four deposits are the following :
Track 1 : proteins from the median sericigenic glands of a non-transgenic silk worm;
Track 2 : proteins from the median sericigenic glands of a transgenic silk
worm heterozygotic for the transgene contained in the sequence SEQ ID M>i;
Track 3 : proteins from the posterior sericigenic glands of a non-transgenic
silk worm; Track 4 : proteins from the posterior sericigenic glands of a transgenic silk
worm heterozygotic for the transgene contained in the sequence
SEQID№l. The filter represented in Figure 6A shows bands of an intensity comparable

from one track to another. It can be concluded that equivalent quantities of proteins have been deposited on the SDS-PAGE gel. The filter shown also comprises a visible size marker in the left part of the filter. The same filter, subjected to treatment with antibody as described hereinbefore is represented in Figure 6B. It is observed that the Ds-Red protein is present in considerable quantity in the posterior sericigenic glands. The Ds-Red protein is also present in the median sericigenic glands, but to a lesser degree. The inventors believe that this presence is due to the migration of the Ds-Red protein secreted by the posterior sericigenic cells in the median sericigenic glands and not to a synthesis of the Ds-Red protein by the median sericigenic glands. Example 5 : Accumulation of the Ds-Red protein in the silk worms rendered transgenic using the Pigfhx(sp*)DsRed-(3xP3 GFP) vector derived from the piggyBac transposon comprising the sequence SEQ ID № 1.
The test for detecting the presence of the Ds-Red protein was done according to the protocol described hereinbefore. As can be seen in Figure 7, the silk fibers have a fluorescence corresponding to the presence of the Ds-Red protein. From this it can be deduced that the nucleic acids according to the invention drives the secretion of a polypeptide of interest in the silk of a lepidopteran.
This result is confirmed by the Western blot represented in Figure 8. An SDS-PAGE gel (14 %; Tris - glycine; 0.1 % SDS) was transferred to a nitrocellulose filter and stained with ponceau red.
The nitrocellulose filter is shown in Figure 8A. This same filter was then incubated with a primary polyclonal anti-Ds-Red antibody and then using a secondary goat anti-rabbit antibody conjugated with horseradish peroxidase. The result is shown in Figure 6B. Bands detected by chemoluminescence can be seen, corresponding to the presence of the Ds-Red protein. Nine deposits of proteins from cocoons of the silkworm are visible on the filter shown in Figure 8A and 8B. The proteins were extracted using the protocol described hereinbefore. These are :
Tracks Cl and C2 : proteins taken from a non-transgenic silk worm cocoon;
Tracks Tl and T2 : proteins taken from a transgenic silk worm cocoon,
heterozygous for the transgene described in the sequence SEQ ID № 1.
The six first deposits, starting from the size marker, shown at the left of the
filter, correspond to proteins from a cocoon of a silk worm from which the exterior
envelope, called the blaze, has been removed. The three latter deposits correspond to
the proteins obtained from the rinsing of the blaze of the cocoon of a silk worm using
water. The filter represented in Figure 8A allows the observation that the quantities of
proteins deposited initially on the SDS-PAGE gel are equivalent. The same filter,
subjected to the treatment described hereinbefore is represented in Figure 8B. It can be
seen that the Ds-Red protein is present in considerable quantity in the fraction obtained

from the rinsing of the transgenic silk worm cocoon blaze. From this it can be deduced that the nucleic acids according to the invention drives the secretion of a polypeptide of interest in the silk of a lepidopteran.
Example 6 : Accumulation of the Ds-Red protein on the surface of the silk fiber of the worm rendered transgenic using the construction described in Example 1.
Figure 9 represents the presence of the Ds-Red protein at the surface of the fiber observed using scanning electron microscopy; The protein appears in the form of globular particles of variable size (depending on the number of aggregates) coating the fiber.
After wetting a transgenic cocoon in an aqueous solution, a concentration measurement (using the classical Bradford method) of the proteins from the corresponding supernatant showed the presence of approximately 100 micrograms of Ds-Red per cocoon.
It can be seen in Figure 9, that the non-fibrous Ds-red protein is sent to the exterior of the silk fiber in the course of secretion. This phenomenon facilitates extraction of the non-fibrous proteins of interest. An identical conclusion can be drawn in the case, for example, of the production of human interleukin-2, as will be described in Example 7.
Example 7 : Production of human IL-2 Production of a transgenic silk worm secreting human interleukin-2 (IL-2)
The gene coding for IL-2 was substituted for the gene coding for the Ds-Red protein in the integration vector described in Example 1. Selection of the transgenic animals
Selection of the transgenic animals is done in virtue of the early expression of the 3xP3-GFP gene in the eyes of the animal. Figure 10 represents the detection of the IL-2 protein placed under the control of a nucleic acid according to the invention in the cocoons of the transgenic heterozygous silk worm;
Tests for detecting IL-2 in the sericigenic glands or in the cocoons of transgenic animals
Detection of protein extracts obtained from sericigenic glands or from cocoons, is done by the Western blot method using a double - antibody: a polyclonal human anti-lL2 antibody and a goat anti-rabbit antibody conjugated with peroxidase (Biorad, reference 170-6515). The test for detecting exogenous proteins by Western blot consists of the steps of electrophoresis, transfer and staining of the proteins, development of the membranes identical to those steps described in the materials and method part relative to the Ds-Red protein.
Only the step corresponding to the extraction of protein samples is different from that described regarding the Ds-Red protein and will therefore be described more

specifically in the following. Extraction of the protein samples
Starting with a 141 mg cocoon :
- 46 mg of the cocoon are subjected to total extraction (sample № 1)
- 19 mg constitute the blaze (the exterior envelope of the cocoon) (sample N» 2)
- 76 mg of the cocoon are subjected to degumming (elimination of the sericins,
proteins coating the fiber), resulting in two fractions :
- one insoluble (sample N° 3)
- the other soluble (sample Jsfa 4).
Treatment of the samples :
- Sample N° 1 : the cocoon, after fragmentation into small pieces and being
dissolved 1 h at ambient temperature in LiSCN 10 M (250 jjL) then, after
dilution in 4/5 Tris 10 mM buffer-2% SDS-5 % p-mercaptoethanol, it is
incubated over night at ambient temperature and 30 minutes at 37 °C.
- Sample N° 2 : the blaze is dissolved for 1 h at ambient temperature in LiSCN 10
M (200 μL) and incubated over night at ambient temperature and at 37 °C for
thirty minutes then the sample is diluted in 4/5 Tris 10 mM buffer- 2 % SDS -
5 % p-mercaptoethanol.
- Samples 3 and 4 : the cocoon is incubated for 1 h at 100 °C in 2 ml of soapy
water (7 g/L) (elimination of the sericins). The soluble fraction (supernatant
containing the sericins) is incubated over night at ambient temperature (sample
N° 4). The insoluble fraction (containing the fibroin) is dissolved over night at
ambient temperature in LiSCN 10M (500 (μL) then the sample is diluted in 4/5
of Tris 10 mM buffer - 2 % SDS - 5 % p-mercaptoethanol. The soluble fraction
(supernatant containing the sericins) is incubated over night at ambient
temperature (sample № 4). The insoluble fraction (containing the fibroin) is
dissolved over night at ambient temperature in LiSCN 10 (500 jaL) then the
sample is diluted in 4/1 Tris 10 mM buffer - 2 % SDS - 5 % P-mercaptoethanol
(sample №3).
Prior to deposit, the samples containing the protein extracts are denatured in Laemmli 5x buffer for 5 minutes at 100 °C.
The following steps are identical to those described for Ds-Red.
The Western blot represented in Figure 10 shows the presence of the IL-2 protein regardless of the sample type being considered.
- Track 1 : proteins obtained from the total extraction of the cocoon (sample
NO 1);
- Track 3 : proteins obtained from the extraction of the blaze (sample N° 2);
- Track 4 : proteins obtained from the insoluble fraction (fibroin) of the

cocoon (sample № 3);
- Track 5 : proteins obtained from the soluble fraction (sericins) of the cocoon
(sample № 4);
- Track 2 : proteins obtained from the total extraction of the control cocoon
(negative control) treated under the same conditions as sample № 1;
- Track S : deposit of 10 ng of a standard solution of human IL-2.
With reference to the track it is possible to have a semi-quantitative estimation of the quantity of IL-2 produced per cocoon. This latter according to the transgenic line analyzed varies from 1 to 10 μg of IL-2 produced per cocoon. This example clearly demonstrates the validity of the invention in the case of a molecule of medical interest, human interleukin, is produced. Moreover, the non-fibrous protein of biomedical interest can be easily purified because it is sent to the exterior of the silk fiber at the time of its synthesis, as has already been demonstrated in Example 6, in the case of the Ds-Red protein.
Example 8 : Production of fibroin H of Galleria mellonella Production of a transgenic silk worm secreting fibroin H of Galleria mellonella
The gene coding for fibroin H of Galleria mellonella was substituted for the gene coding for the Ds-Red protein in the integration vector described in Example 1. Figure 11 represents the detection of the fibroin H protein of Galleria mellonella placed under the control of a nucleic acid according to the invention in the sericigenic glands (A) and the cocoons (B) of heterozygous transgenic silk worms, Tests for detection of fibroin H of Galleria mellonella in the sericigenic glands or the cocoons of transgenic animals
Detection of protein extracts obtained from sericigenic glands or from cocoons, is done by the Western blot method using a double - antibody: a polyclonal human anti-FibHGm antibody and a goat anti-rabbit antibody conjugated with peroxidase (Biorad, reference 170-6515). The test for detecting exogenous proteins by Western blot consists of the steps of electrophoresis, transfer and staining of the proteins, development of the membranes identical to those steps described in the materials and method part relative to the Ds-Red protein. Only the step corresponding to the extraction of protein samples is different from that described regarding the Ds-Red protein and will therefore be described more specifically in the following. Extraction and treatment of the protein samples
The pair of sericigenic glands (secretor separated from the reservoir) taken from transgenic animals at the fifth larval stage is transferred into a microtube, crushed and incubated in 500 μL of LiSCN for 20 minutes at ambient temperature and then 1 hour at 60 °C. The sample is then diluted in 4/5 of Tris 10 mM buffer - 2 % SDS - 5 % (5-mercaptoethanol.

The cocoon (20 mg) after fragmentation into small pieces is dissolved for 30 minutes at 65 °C in LiSCN 10M (250 μL) and then diluted in 4/5 Tris 10 mM buffer-2% SDS - 5 % p-mercaptoethanol.
Prior to deposit, the samples containing the protein extracts are denatured in Laemmli 5x buffer for 5 minutes at 100 °C. The following steps are identical to those described for Ds-Red.
- The Western blot represented in Figure 11 shows the presence of the FibHGm
(size : 115 KDa) in the sericigenic gland (reservoir and secretor) and the cocoon
of the transgenic individual, while this protein is absent in the control samples
obtained from non-transgenic animals, samples that were treated under identical
conditions as those described hereinbefore.
Track 1 : proteins obtained from the extraction of the secretor part of the
sericigenic gland of a transgenic animal;
Track 2 : proteins obtained from the extraction of the reservoir part of the
sericigenic gland of a transgenic animal;
Track 3 : proteins obtained from the total extraction of a cocoon obtained from
a transgenic animal;
Track 1* : proteins obtained from the extraction of the secretor part of the
sericigenic gland of a control animal;
Track 2* : proteins obtained from the extraction of the reservoir part of the
sericigenic gland of a control animal;
Track 3* : proteins obtained from the total extraction of a cocoon obtained from
a control animal.
This example clearly demonstrates the validity of the invention for the production of novel biomaterials such as the silk mixtures having novel mechanical properties of strength and elasticity.
Example 9 : Protocol for the construction of a vector comprising a nucleic acid directing the expression of an RNA reducing the synthesis of fibroin H.
In order to inhibit fibroin H, a transgenesis piggyBac vector as described in Example 1, comprising two fragments of the gene of interest placed in the reverse -repeat direction is produced. The sense - anti-sense sequences derived from the fibroin H gene are spaced by a neutral stabilizing sequence and under the control of the fibrohexamerin promoter (Figure 12). This vector drives the production of double-stranded sRNA comprised of a single chain that folds in order to form a secondary structure, the so-called "hairpin", which targets a moiety of 465 bps of repeat sequences located in exon 2 of the fibroin H gene (Figure 12). These sequences are formed alternatively from repeat and amorphous domains. As can be seen in Figure 12, the size of the fibroin H gene is 17 kbp. The coding sequences are comprise of two

exons : one 67 bps and the other 15,750 bp. This gene is characterized by extremely xspeated sequences in exon 2. Each repeat region is comprised of sub-domains of 208 of) organized in repeat Ua and Ub motifs. The position of the 465 bps fragment used for targeting the mRNA is represented by the arrows. The repetitions of the mRNA produced after transcription are diagrammatically represented in gray. (Zhou et al., 2000 Fine organization of Bombyx mori fibroin heavy chain gene. Nucleic Acids Res 28,2413-2419).
Co-injected with the assistant vector (helper) described in the "general materials and method part", this construction has allowed the establishment of transgenic silk worm lines for which the quantity of fibroin H produced in the cocoons has been measured.
As shown in Figure 1 (histogram), the RNA mechanism produces a measurable effect, variable along the transgenic lines, on the secretion of fibroin H.
The cocoons spun by the transgenic silkworms contain up to 20 % less fibroin H relative to control cocoons.



WE CLAIM:
1. A nucleic acid directing the expression of a protein of interest specifically
in the cells of the posterior sericigenic glands of Bombyx mori, wherein said
nucleic acid comprises, from the 5' terminus to the 3' terminus, (i) a regulatory
region comprising regulatory signals for the expression of a polynucleotide of
interest specifically in said sericigenic cells and (ii) a region coding for a
modified signal peptide placed under the control of said regulatory region, said
nucleic acid having at least 90% identity in nucleotides with the polynucleotide
running from nucleotide 1150 to nucleotide 2026 of the sequence SEQ ID NO:
1 and in which the trinucleotide running from the nucleotide 1486 to
nucleotide 1488 of sequence SEQ ID NO: 1 codes for an amino acid chosen
from the group comprising alanine, isoleucine and leucine.
2. The nucleic acid as claimed in claim 1, wherein, said nucleic acid comprises, from the 5' terminus to the 3' terminus, (i) a regulatory region comprising regulatory signals for the expression of a polynucleotide of interest specifically in said sericigenic cells and (ii) a region coding for a modified signal peptide, placed under the control of said regulatory region, said nucleic acid comprising, from the 5' terminus to the 3' terminus, the polynucleotide running from nucleotide 1150 to nucleotide 2026 of sequence SEQ ID NO: 1 and in which the trinucleotide running from the nucleotide 1486 to the nucleotide 1488 of sequence SEQ ID NO: 1 codes for an amino acid chosen from the group comprising alanine, isoleucine, and leucine.
3. The nucleic acid as claimed in claim 1, wherein, said nucleic acid comprises, from the 5' terminus to the 3' terminus, (i) a regulatory region comprising regulatory signals for the expression of a polynucleotide of interest specifically in said sericigenic cells and (ii) a region coding for a modified signal peptide, placed under the control of said regulatory region, said nucleic acid comprising, from the 5' terminus to the 3' terminus, the

polynucleotide running from nucleotide 1 to nucleotide 2026 of sequence SEQ ID NO: 1 and in which the trinucleotide running from the nucleotide 1486 to the nucleotide 1488 of sequence SEQ ID NO: 1 codes for an amino acid chosen from the group comprising alanine, isoleucine, and leucine.
4. A nucleic acid consisting of a nucleic acid as claimed in one of claims 1 to 3, which has been modified by the introduction of at least one and not more than four copies of a polynucleotide of sequence SEQ ID NO: 2 between nucleotides 1 and 1379 of sequence SEQ ID NO: 1.
5. A nucleic acid having a sequence that is complementary to a nucleic acid as claimed in one of claims 1 to 4.
6. An expression cassette comprising a polynucleotide of interest placed under the control of a nucleic acid as claimed in one of claims 1 to 5.
7. The expression cassette as claimed in claim 6, wherein the polynucleotide of interest codes for a polypeptide.
8. The expression cassette as claimed in claim 6, wherein the polynucleotide of interest codes for a fusion polypeptide comprising, from the N-terminus end to the C-terminus end:
- at least the 172 N-terminus amino acids of the fibroin L polypeptide
sequence, and
- a polypeptide of interest.
9. The expression cassette as claimed in claim 7 or 8, wherein the polypeptide
of interest is chosen from among the following proteins:
- spider spidroin;
- fibroin of animals of the genus Galleria, and

- human interleukin-2.

10. A recombinant integrative vector comprising a nucleic acid as claimed in claims 1 to 5 or an expression cassette as claimed in one of claims 6 to 9.
11. A recombinant cloning vector comprising a nucleotide sequence of a recombinant integration vector as claimed in claim 10.
12. The recombinant integrative vector as claimed in claim 10, wherein it is the Pigfhx(sp*)DsRed-(3xP3-GFP) deposited on February 20, 2003 in the Collection Nationale de Cultures de Micro-organisms (CNCM) of the Institut Pasteur under CNCM registration number 1-2975.

Documents:

4054-DELNP-2005-Abstract-(30-01-2009).pdf

4054-delnp-2005-abstract.pdf

4054-DELNP-2005-Claims-(30-01-2009).pdf

4054-delnp-2005-claims.pdf

4054-delnp-2005-correspondence others.pdf

4054-DELNP-2005-Correspondence-Others-(30-01-2009).pdf

4054-delnp-2005-description (complete).pdf

4054-delnp-2005-drawings.pdf

4054-DELNP-2005-Form-1-(30-01-2009).pdf

4054-delnp-2005-form-1.pdf

4054-delnp-2005-form-18.pdf

4054-DELNP-2005-Form-2-(30-01-2009).pdf

4054-delnp-2005-form-2.pdf

4054-delnp-2005-form-3.pdf

4054-delnp-2005-form-5.pdf

4054-DELNP-2005-GPA-(30-01-2009).pdf

4054-delnp-2005-gpa.pdf

4054-delnp-2005-pct-308.pdf

4054-DELNP-2005-Petition-137-(30-01-2009).pdf


Patent Number 228680
Indian Patent Application Number 4054/DELNP/2005
PG Journal Number 13/2009
Publication Date 27-Mar-2009
Grant Date 06-Feb-2009
Date of Filing 09-Sep-2005
Name of Patentee CENTRE NATIONAL DE LA RECHERCHE SCIENTIFIQUE [CNRS]
Applicant Address 3, RUE MICHEL-ANGE, 75794 PARIS CEDEX 16, FRANCE.
Inventors:
# Inventor's Name Inventor's Address
1 BENEDICTE DURAND 11 RUE MAGNEVAL, 69001 LYON, FRANCE.
2 ANN-MARIE GRENIER 6 RUE DES MESANGES, 69680 CHASSIEU, FRANCE.
3 BEATRICE HORARD 4 ALLEE DU PARC, 69003 LYON, FRANCE.
4 GERARD CHAVANCY 2 AVENUE ROBERTO ROSSELLINI, 69100 VILLEURBANNE, FRANCE.
5 PIERRE COUBLE 203 AVENUE FLEMING, 69300 CALUIRE, FRANCE.
6 ERIC JULIEN 20 AVENUE FOUCAUD, 87000 LIMOGES, FRANCE.
7 BERNARD MAUCHAMP LES CABANES, 01390 MIONNAY, FRANCE.
8 PASCALE NONY 4 AVENUE DES FRERES LUMIERE, 69008 LYON, FRANCE.
9 JEAN-CLAUDE PRUDHOMME 70B RUE PIERRE BRUNIER, 69300 CALUIRE, FRANCE.
10 CORINNE ROYER 96 CHEMIN DES FONTS, 69110 ST-FOY-LES-LYON, FRANCE.
PCT International Classification Number C12Q
PCT International Application Number PCT/FR2004/050107
PCT International Filing date 2004-03-12
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 03/03137 2003-03-13 France