Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1999-08-10
2001-04-17
Brusca, John S. (Department: 1631)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S173300, C435S320100, C536S024200, C536S023100
Reexamination Certificate
active
06218123
ABSTRACT:
1.0 FIELD OF THE INVENTION
The present invention relates to a complex cDNA library generated from eukaryotic cells and methods for making such a library. More specifically, the invention uses recombinant constructs that randomly insert into the genome to facilitate the expression of nuclear genes as fusion transcripts. The invention further allows one to specifically subclone the corresponding fusion transcripts into a highly complex cDNA library. The library is easily characterized by molecular analysis techniques, and individual clones can be directly sequenced to generate a sequence database of the cell-derived portion of the fusion transcripts.
2.0 BACKGROUND OF THE INVENTION
The Human Genome Project is currently approaching the sequencing phase of the human genome and the completion of this milestone is expected in the year 2005. The hope is that at the conclusion of the sequencing phase, a comprehensive representation of the human genome will be available for biomedical analysis. However, the resulting sequence data from the human genome project will typically correspond to human genomic sequence, and the actual genes represented in the genomic sequence might not be obvious even with the use of sophisticated computer assisted exon identification programs. The availability of cDNA information will therefore significantly contribute to the value of the sequenced human genome since they directly indicate the presence of transcribed sequences. Thus, the sequencing of cDNA libraries to obtain expressed sequence tags or ESTs that identify exons expressed within a given tissue, cell, or cell line is currently in progress. As a consequence of these efforts, a large number of EST sequences are presently compiled in public and privately held databases. However, the present EST paradigm is inherently limited by the levels and extent of mRNA production within a given cell. A related problem is the lack of cDNA sources from specific tissue and developmental expression profiles. In addition, some genes are typically only active under certain physiological conditions or are generally expressed at levels below or near the threshold necessary for cDNA cloning and detection and are therefore not effectively represented in current cDNA libraries.
Researchers have partially addressed these issues by using phage vectors to clone genomic sequences such that internal exons are trapped (Nehls, et al., 1994, Current Biology, 4(1):983-989, and Nehls, et al., 1994, Oncogene, 9:2169-2175). However, such libraries require the random cloning of genomic DNA into a suitable cloning vector in vitro, followed by reintroduction of the cloned DNA in vivo in order to express and splice the cloned genes prior to producing the cDNA library. Additionally, such methods are limited to “trapping” genes having internal exons.
3.0 SUMMARY OF THE INVENTION
The present invention describes methods for constructing complex cDNA libraries from gene trapped eukaryotic cells. Although the presently described libraries can be constructed from virtually any cell that is naturally capable of splicing nuclear mRNA, animal cells, and particularly mammalian cells, are of particular interest.
Rapid production and sequencing of such normalized gene trapped sequence (GTS) libraries greatly facilitates gene identification and complements current sequencing efforts such as, for example, the Human Genome Project. Accordingly, one embodiment of the present invention is directed to cDNA libraries that provide a normalized representation of the genes present within a given cell, cell line, tissue, plant, or animal. In a preferred embodiment, the GTS libraries are produced by a method that does not include a period of selective culture that enriches the population of eucaryotic cells that incorporate the exogenously introduced gene trap construct.
Accordingly, one embodiment of the present invention is a collection of individually isolated and identified human cDNA sequences that collectively comprise at least one sequence representative of each of the following categories: G-protein coupled recpetors, G proteins, cytoskeletal proteins, protein kinases, steroid response element binding proteins, and tumor suppressor proteins.
The presently described methods for generating such libraries can be used to produce normalized (or equalized) cDNA libraries using mRNA obtained from virtually any eukaryotic cell. Using the described methods, each cell or cell clone is engineered to express a gene, or part of a gene (e.g., sequence), under the control of a promoter that has been nonspecifically, or essentially randomly, integrated into the genome of the target cell. For the purposes of the present invention, the term “nonspecifically integrated” shall mean that a polynucleotide has not specifically integrated into a predefined target sequence, or has not been directed to a particular region of the host cell chromosome by the incorporation of one or more regions of flanking homologous “targeting” DNA.
Given that the production of the mRNA pool that is used to produce the described cDNA libraries is mediated by an exogenously added promoter, the presently described procedures allow one to express a higher percentage of the genes collectively present within the target cell population. An additional feature of the fact that the exogenous promoter element is nonspecifically, or even randomly, integrated into the genome of each cell is that the exogenous promoter more-or-less uniformly directs the expression of the mRNA that is preferentially used to generate the cDNA within the library. The uniformity of this expression effectively “normalizes” the relative percentages of the various cDNAs that are incorporated into the described libraries. In essence, the cDNAs in the library are related by the common promoter element. This feature significantly reduces the burden of sequencing duplicative cDNAs that are typically over represented in conventional cDNA libraries because of differential expression levels within the cell.
A particularly useful feature of the presently described procedure, is that genes that are normally not expressed in the target cell, or expressed at low levels, (i.e., effectively undetectable using conventional methods of generating cDNA), are expressed at levels that allow cDNA production and cloning.
REFERENCES:
patent: 5506126 (1996-04-01), Seed et al.
patent: 5556772 (1996-09-01), Sorge et al.
patent: 5646009 (1997-07-01), Rhoads et al.
patent: WO 98/14614 (1998-04-01), None
patent: WO 99/50426 (1999-10-01), None
Rossouw CM et al, “DNA sequences in the first intron of the huyman pro-alpha 1(I) collagen gene enhance trancription”, Journal of Biological Chemistry, Nov. 5, 1987, vol. 262, No. 31, pp. 15151-15157.*
Sasaki K et al, “Cloning and expression of a complementary DNA encoding a bovine adrenal angiotensin II type-1 receptor”, Nature, vol. 351, May 16, 1991, pp. 230-233.*
Yin Jingwen et al, “Stable transfection of Acanthamoeba”, Can. J. Microbiol, vol. 43, 1997, pp. 239-244.*
Nehls et al., “Exon amplification from complete libraries of genomic DNA using a novel phage vector with automatic plasmid excision facility: application to the mouse neurofibromatosis-1 locus,”Oncogene, 9:2169-2175 (1994).
Nehls et al., “The sequence complexity of exons trapped from the mouse genome,”Current Biology, 4(11):983-989 (1994).
Yoshida et al., “A new strategy of gene trapping in ES cells using 3′RACE,”Transgenic Research, 4:277-287 (1995).
Zambrowicz et al., “Disruption and sequence identification of 2,000 genes in mouse embryonic stem cells,”Nature, 392:608-611 (1998).
Zambrowicz et al., “Comprehensive mammalian genetics: history and future prospects of gene trapping in the mouse,”Int. J. Dev. Biol., 42:1025-1036 (1998).
Friedrich Glenn
Nehls Michael
Ruley H. Earl
Sands Arthur T.
Wattler Sigrid
Brusca John S.
Finnegan Henderson Farabow Garrett & Dunner L.L.P.
Lexicon Genetics Incorporated
Siu Stephen
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