Patent ID: 6010908
Filing Date: 2000-01-04
Classification: A01K,A61K,C07K,C12N

Abstract:
A method for in vitro homologous replacement, in target cells, of an exon carrying an endogenous targeted mutant DNA sequence of a gene for Fanconi's anemia, thalassaemias, cystic fibrosis, sickle cell anemia, retinitis pigmentosa, xeroderma pigmentosum, ataxia telangiectasia, Bloom's syndrome, retinoblastoma, Duchenne's muscular dystrophy, or Tay-Sachs disease, with an exogenous replacement DNA fragment comprising 3' and 5' flanking intronic sequences homologous to 3' and 5' flanking sequences of the mutant DNA sequence, said intronic sequences adjacent to an exon carrying a silent mutation that gives rise to a novel restriction enzyme cleavage site not present in the exon carrying the targeted mutant DNA sequence, and the sequence to replace the targeted mutant DNA sequence, said method comprising steps:(a) identifying the exon containing the targeted mutant DNA sequence to be replaced and 3' and 5' flanking intronic sequences adjacent to said exon;(b) generating the exogenous replacement DNA fragment comprising homologous 3' and 5' flanking intronic sequences adjacent to the exon having the sequence to replace the targeted mutant DNA sequence and carrying a silent mutation that gives rise to a novel restriction enzyme cleavage site not present in the exon carrying the targeted mutant DNA sequence;wherein total size of the replacement DNA fragment is from 1 to 2000 bases;wherein the replacement DNA fragment is a single stranded DNA generated from a double stranded fragment by denaturation or by separation of a biotin labeled complementary strand from a strand comprising the replacement fragment;wherein the double stranded fragment is comprised of two complementary DNA strands each of which is able to replace one strand of the targeted genomic DNA;(c) transfecting or delivering the exogenous replacement DNA fragment of step (b) into the target cells containing the targeted mutant DNA sequence, wherein the transfection or the fragment delivery is accomplished by incubating the replacement DNA fragment of step (b) in a transfection cocktail, forming a transfection mix, introducing the incubated transfection mix to the cells of step (c) grown to 70-90% confluence, and incubating the transfection mix in the presence of the cells at about 32 to 40.degree. C. temperature;(d) propagating the incubated cells in a fresh growth medium for about two days to about two weeks; and(f) determining the extent of homologous replacement by PCR identification of cells within the total cell population which have replaced the endogenous targeted mutant DNA with the exogenous replacement DNA fragment at a target genomic locus.