Vectors for making genomic modifications
View Patent ↗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 genome 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 retroviral vector for replacing the 3′ end of an animal cell transcript with a foreign exon, comprising:
a) a foreign exon;
b) a splice acceptor operatively positioned 5′ to said foreign exon;
c) a polyadenylation site operatively positioned 3′ to said foreign exon;
d) a first site-specific recombinase site upstream of the splice acceptor; and
e) a second site-specific recombinase site downstream of the splice acceptor;
wherein said vector does not comprise a promoter element operatively positioned relative to the foreign exon.
2. The vector of claim 1 , wherein the second recombinase site is downstream of the polyadenylation site.
3. The vector of claim 1 , wherein the first and second recombinase sites are selected from loxP and frt.
4. The vector of claim 1 , wherein the foreign exon is a selectable marker.
5. A method of producing a collection of mutated animal cells, comprising
exposing a group of animal cells in vitro to the vector of claim 1 under conditions allowing stable integration of the vector into an animal cell genome; and
selecting cells that express the product encoded by the foreign exon.