Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1997-10-02
2001-03-27
Guzo, David (Department: 1636)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S320100, C435S325000, C435S456000, C536S023100, C536S024100
Reexamination Certificate
active
06207371
ABSTRACT:
1.0. FIELD OF THE INVENTION
The invention relates to an indexed library of genetically altered cells and methods of organizing the cells into an easily manipulated and characterized Library. The invention also relates to methods of making the library, vectors for making insertion mutations in genes, methods of gathering sequence information from each member clone of the Library, and methods of isolating a particular clone of interest from the Library.
2.0. BACKGROUND OF THE INVENTION
The general technologies of targeting mutations into the genome of cells, and the process of generating mouse lines from genetically altered embryonic stem (ES) cells with specific genetic lesions are well known (Bradley, 1991, Cur. Opin. Biotech. 2:823-829). A random method of generating genetic lesions in cells (called gene, or promoter, trapping) has been developed in parallel with the targeted methods of genetic mutation (Allen et al., 1988 Nature 333(6176):852-855; Brenner et al., 1989, Proc. Natl. Acad. Sci. U.S.A. 86(14):5517-5521; Chang et al., 1993, Virology 193(2):737-747; Friedrich and Soriano, 1993, Insertional mutagenesis by retroviruses and promoter traps in embryonic stem cells, p. 681-701. In Methods Enzymol., vol. 225., P. M. Wassarman and M. L. DePamphilis (ed.), Academic Press, Inc., San Diego; Friedrich and Soriano, 1991, Genes Dev. 5(9):1513-1523; Gossler et al., 1989, Science 244(4903):463-465; Kerr et al., 1989, Cold Spring Harb. Symp. Quant. Biol. 2:767-776; Reddy et al., 1991, J Virol. 65(3):1507-1515; Reddy et al., 1992, Proc. Natl. Acad. Sci. U.S.A. 89(15):6721-6725; Skarnes et al., 1992, Genes Dev. 6(6):903-918; von Melchner and Ruley, 1989, J. Virol. 63(8):3227-3233; Yoshida et al., 1995, Transgen. Res. 4:277-287). Gene trapping provides a means to create a collection of random mutations by inserting fragments of DNA into transcribed genes. Insertions into transcribed genes are selected over the background of total insertions since the mutagenic DNA encodes an antibiotic resistance gene or some other selectable marker. The selectable marker lacks its own promoter and enhancer and must be expressed by the endogenous sequences that flank the marker after it has integrated. Using this approach, transcription of the selectable marker is activated and the cell gene is concurrently mutated. This type of strict selection makes it possible to easily isolate thousands of ES cell colonies, each with a unique mutagenic insertion.
Collecting mutants on a large-scale has been a powerful genetic technique commonly used for organisms which are more amenable to such analysis than mammals. These organisms, such as
Drosophila melanogastor,
yeast
Saccharomyces cerevisiae,
and plants such as
Arabadopsis thalia
are small, have short generation times and small genomes (Bellen et al., 1989, Genes Dev. 3(9):1288-1300; Bier et al., 1989, Genes Dev. 3(9):1273-1287; Hope, 1991, Develop. 113(2):399-408. These features allow an investigator to rear many thousands or millions of different mutant strains without requiring unmanageable resources. However, these type of organisms have only limited value in the study of biology relevant to human physiology and health. It is therefore important to have the power of large-scale genetic analysis available for the study of a mammalian species that can aid in the study of human disease. Given that the entire human genome is presently being sequenced, the comprehensive genetic analysis of a related mammalian species will provide a means to determine the function of genes cloned from the human genome. At present, rodents, and particularly mice, provide the best model for genetic manipulation and analysis of mammalian physiology.
Gene trapping has been used as an analytical tool to identify genes and regulatory regions in a variety of animal cell types. One system that has proved particularly useful is based on the use of ROSA (reverse orientation splice acceptor) retroviral vectors (Friedrich and Soriano, 1991 and 1993).
The ROSA system can generate mutations that result in a detectable homozygous phenotype with a high frequency. About 50% of all the insertions caused embryonic lethality. The specifically mutated genes may easily be cloned since the gene trapping event produces a fusion transcript. This fusion transcript has trapped exon sequences appended to the sequences of the selectable marker allowing the latter to be used as a tag in polymerase chain reaction (PCR)-based protocols, or by simple cDNA cloning. Examples of genes isolated by these methods include a transcription factor related to human TEF-1 (transcription enhancer factor-1) which is required in the development of the heart (Chen et al., 1994, Genes Devel. 8:2293-2301. Another (spock), is distantly related to yeast genes encoding secretion proteins and is important during gastrulation.
The above experiments have established that the ROSA system is an effective analytical tool for genetic analysis in mammals. However, the structure of many ROSA vectors selects for the “trapping” of 5
40
exons which, in many cases, do not encode proteins. Such a result is adequate where one wishes to identify and eventually clone control (i.e., promoter or enhancer) sequences, but is not optimal where the generation of insertion-inactivated null mutations is desired, and relevant coding sequence is needed. Thus, the construction of large-scale mutant (preferably null mutant) libraries requires the use of vectors that have been designed to select for insertion events that have occurred within the coding region of the mutated genes as well as vectors that are not limited to detecting insertions into expressed genes.
3.0. SUMMARY OF THE INVENTION
An object of the present invention is to provide a set of genetically altered cells (the ‘Library’). The genetic alterations are of sufficient randomness and frequency such that the combined population of cells in the Library represent mutations in essentially every gene found in the cell's genome. The Library is used as a source for obtaining specifically mutated cells, cell lines derived from the individually mutated cells, and cells for use in the production of transgenic non-human animals.
A further object is to provide the vectors, both DNA and retroviral based, that may be used to generate the Library. Typically, at least two distinct vector designs will be used in order to mutate genes that are actively expressed in the target cell, and genes that are not expressed in the target cell. Combining the mutant cells obtained using both types of vectors best ensures that the Library provides a comprehensive set of gene mutations.
A particularly useful vector class contemplated by the present invention includes a vector for inserting foreign exons into animal cell transcripts that comprises a selectable marker, a promoter element operatively positioned 5′ to the selectable marker, a splice donor site operatively positioned 3′ to the selectable marker, and a second mutagenic foreign polynucleotide sequence located upstream from the promoter element that disrupts, or otherwise “poisons”, the splicing or read-through expression of the endogenous cellular transcript. Typically, the mutagenic foreign polynucleotide sequence may incorporate a polyadenylation (pA) site, a nested set of stop codons in each of the three reading frames, splice acceptor and splice donor sequences in operable combination, a mutagenic exon, or any mixture of mutagenic features that effectively prevent the expression of the cellular gene. For example, a polyadenylation sequence may be incorporated in addition to or in lieu of the splice donor sequence. A preferred organization for the mutagenic polynucleotide sequence comprises a polyadenylation site positioned upstream from a selectable marker which is in turn located upstream from a splice acceptor sequence. Preferably, such a vector does not comprise a transcription terminator or polyadenylation site operatively positioned relative to the coding region of the selectable marker, and shall not comprise a splice acceptor sit
Bradley Allan
Friedrich Glenn A.
Sands Arthur T.
Zambrowicz Brian
Finnegan Henderson Farabow Garrett & Dunner
Guzo David
Lexicon Genetics Incorporated
Sandals William
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