Chemistry: molecular biology and microbiology – Animal cell – per se ; composition thereof; process of...
Reexamination Certificate
2000-03-29
2003-02-11
Nguyen, Dave T. (Department: 1632)
Chemistry: molecular biology and microbiology
Animal cell, per se ; composition thereof; process of...
C435S320100, C435S069100, C435S091400, C435S455000, C536S023100, C536S024100
Reexamination Certificate
active
06518064
ABSTRACT:
BACKGROUND OF THE INVENTION
A variety of systems, both prokaryotic and eukaryotic, are available for producing therapeutically important proteins. The use of prokaryotic systems is, however, limited, since prokaryotic cells cannot carry out the post-translational modifications that are often essential for the function of eukaryotic proteins. In addition, secretion of eukaryotic proteins is often inefficient in prokaryotic systems, and folding of the produced protein is absent or incorrect. A number of eukaryotic systems have also been developed. Such eukaryotic expression systems can successfully express native and active proteins that are not capable of being produced in prokaryotic systems. Mammalian systems are however expensive and zoonoses, in particular prion diseases, may be transmitted by use of mammalian products (e.g., Fetal Calf Serum) in in vitro culture systems. Furthermore, many desirable proteins, such as anti-clotting factors or peptide hormones may have activity in mammalian or vertebrate systems that prevent their expression in these systems. For example, anti-clotting factors produced in large amounts in mammals would likely be fatal to those animals due to lack of clotting, obviating the production of these materials in a mammalian system. Insects systems offer an alternative to mammalian systems. Insect systems are cheaper and are not associated with the same health issues as their mammalian counterparts. However, to date, the available insect systems is limited and they do not allow large scale protein production without cell culture.
Many insects, and in particular Lepidopteran species, are key pests on food and fiber crops worldwide. Field resistance to pesticides presents an increasing challenge to pest control. Furthering the ability to manipulate insect species at the molecular level will become more important to advance our understanding of insect biology and to promote the development of novel field applications. As an example, germ-line transformation can directly contribute to pest control efforts, for instance, through indelible genetic marking of insects produced for Sterile Insect Technique (SIT) programs.
The utility of transposable elements to manipulate an insect genome has been shown in Drosophila with the P element (Rubin and Spradling (1983); Spradling and Rubin (1982)). However, the host range of P is restricted to closely related drosophilids (O'Brochta et al. (1991)
Mol. Gen. Genet.
225:387-394; O'Brochta et al. (1994)
Mol. Gen. Genet.
244:9-14)). The discovery of new elements with broader host ranges such as mariner (Coates et al. (1995)
Mol. Gen. Genet.
249:246-252), Minos (Loukeris et al. (1995)
Science
270:2002-2005), Hermes (O'Brochta et al. (1996)
Insect Biochem. Mol. Biol.
26:739-753; Sarkar et al. (1997)
Genetica
99:15-29; Sarkar et al. (1997)
Insect Biochem. Mol. Biol.
27:359-363), and piggyBac (Loukeris et al., supra) has led to the development of vector systems for transformation of further insect species including the medfly,
Ceratitis capitata
(Loukeris et al., supra), the mosquito
Aedes aegypti
(Jasinskiene et al. (1998)
Proc. Natl. Acad. Sci. USA
95:3743-3747; Coates et al. (1998)
Proc. Natl. Acad. Sci. USA
95:3748-3751) and the domestic silkworm
Bombyx mori
(Toshiki et al. (2000)
Nature Biotech.
18:81-84). Most successful germ-line transformation events have been described for Dipteran species.
There is therefore a need in the art for insect transformation and protein expression systems, for protein production and for other uses, such as insect marking for pest control studies. The present invention addresses these and other needs.
SUMMARY OF THE INVENTION
The present invention provides promoters, expression cassettes, expression vectors and host cells for expressing heterologous proteins in insects. The invention further provides a method for transforming insect cells, in particular from a Lepidopteran, and for obtaining transgenic insects. The methods of the present invention can be used for, for example, to produce a protein of interest or to mark insects using marker proteins.
In one aspect, the present invention is directed to an isolated nucleic acid molecule comprising a promoter sequence having at least 90% identity with the sequence set forth in SEQ ID NO:1. In one embodiment, the promoter has the sequence of SEQ ID NO:1. The present invention further provides an expression cassette comprising the promoter sequence operably linked to a nucleic acid sequence encoding a protein of interest. In some embodiments, the expression cassette further comprises a transcriptional enhancer. In other embodiments, the expression cassette further comprises a regulatory sequence that controls the tissue-specific expression of the protein of interest. In some embodiments, the protein is a fluorescent marker. The fluorescent marker can be, for example, the enhanced green fluorescent protein, derivatives therof or DSred. In another embodiment, the protein of interest is a therapeutic protein. In other embodiments, the nucleic acid sequence is linked to the promoter sequence in an antisense orientation.
The present invention also provides expression vectors and host cells comprising the expression cassette described above. In some embodiments, the expression vector is a transposable element, for example, piggyBac. The piggyBac transposable element of the invention preferably lacks a 1.023 kb fragment from the Open Reading Frame encoding the transposase.
In other aspects, the invention is directed to transgenic insects comprising the expression cassette described above. In some embodiments, the transgenic insect is a Lepidopteran, such as the pink bollworm. In other embodiments, the insect is a Dipteran.
The present invention further provides a method for transforming an insect cell comprising introducing into the cell an expression cassette of the invention. In some embodiments, the cell is a pre-zygotic egg cell. In some embodiments, the pre-zygotic egg cell is allowed to develop into an insect. In other embodiments, the cell is within an insect. The insect cell can be from a Lepidopteran, such as a pink bollworm. In one aspect, the insect cell is cultured to obtain a cell culture, and the protein of interest is purified from the cell culture. In another aspect, the expression cassette comprises a nucleic acid sequence encoding a marker protein.
The present invention further provides a method for producing a protein comprising introducing into an insect cell an expression cassette, allowing the cell to develop into an insect, and purifying the protein of interest from the insect or its progeny.
REFERENCES:
Ngo et al., in: The Protein Folding Problems and Tertiary Structure Prediction, 1994, Merz et al., (ed.), Birkhauser, Boston, MA, pp. 492-495.*
Mikkelsen, TIG, 1993, vol. 9, p. 159.*
Chiu et al. Folding & Design, 1998, vol. 3, No. 3, pp. 223-228.*
Handler, Alfred M., et. al., “The lepiopteran transposon vector, piggyBac, mediates germ-line transformation in the Mediterranean fruit fly” Proc. Natl. Acad. Sci. USA, 1998, 95:7520-7525.
Lobo, N., et al., “Transposition of the piggyBac element in embryos of Drosophila melanogaster, Aedes aegypti and Trichoplusia ni” Mol. Gen. Genet., 1999, 261:803-810.
Farrell, Patrick J., et al., “High-Level Expression of Secreted Glycoproteins in Transformed Lepidopteran Insect Cells Using a Novel Expression Vector” Biotechnol., Bioeng., 1998, 60:656-663.
Mange, Alain, et al., “A Strong Inhibitory Element Down-regulates SRE-stimulated Transcription of the A3 Cytoplasmic Actin Gene of Bombyx mori” J. Mol. Biol., 1997, 265:266-274.
Toshiki, Tamura, et al., “Germline transformation of the silkworm Bombyx mori L. using piggyBac transposon-derived vector” Nat. Biotechnology, 2000, 18:81-84.
Miller Thomas A.
Peloquin John J.
Nguyen Dave T.
Townsend and Townsend / and Crew LLP
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