Indexed library of cells containing genomic modifications and methods of making and utilizing the same
Methods and vectors (both DNA and retroviral) are provided for the construction of a Library of mutated cells. The Library will preferably contain mutations in essentially all genes present in the gene of the cells. The nature of the Library and the vectors allow for methods of screening for mutations in specific genes, and for gathering nucleotide sequence data from each mutated gene to provide a database of tagged gene sequences. Such a database provides a means to access the individual mutant cell clones contained in the Library. The invention includes the described Library, methods of making the same, and vectors used to construct the Library. Methods are also provided for accessing individual parts of the Library either by sequence or by pooling and screening. The invention also provides for the generation of non-human transgenic animals which are mutant for specific genes as isolated and generated from the cells of the Library.
1. A library of cultured eucaryotic cells comprising at least two subpopulations of cells, wherein the at least two subpopulations of cells comprises
(a) a first subpopulation of cells, wherein cells of the first subpopulation have a first vector integrated nonspecifically into their genomes, wherein the first vector mediates the splicing of a foreign exon internal to a cellular transcript, and wherein the first vector comprises:
(i) a foreign exon,
(ii) a splice acceptor site operatively positioned 5′ to said foreign exon, and
(iii) a splice donor site operatively positioned 3′ to said foreign exon, wherein the first vector does not comprise a promoter operatively positioned 5′ to said foreign exon; and
(b) a second subpopulation of cells, wherein cells of the second subpopulation have a second vector integrated nonspecifically into their genomes, wherein the second vector mediates the splicing of a foreign exon 5′ to an exon of a cellular transcript, and wherein the second vector comprises:
(i) a foreign exon,
(ii) a promoter operatively positioned 5′ to said foreign exon,
(iii) a splice donor site operatively positioned 3′ to said foreign exon; and
(iv) a mutagenic foreign polynucleotide sequence located upstream from said promoter.
2. The library of claim 1 wherein said cultured eucaryotic cells are animal cells.
3. The library of claim 2 wherein said cultured eucaryotic cells are mammalian cells.
4. The library of claim 3 wherein said cultured eucaryotic cells are rodent cells.
5. The library of claim 4 wherein said cultured eucaryotic cells are mouse cells.
6. The library of claim 1 , wherein said cultured eucaryotic cells are organized into individual clones.