Efficient combination of large DNA in vitro: in gel site specific recombination (IGSSR) of PAC fragments containing alpha satellite DNA and the human HPRT gene locus.
In an attempt to combine a cloned genomic copy of a selectable gene with different cloned centromeric sequences to develop mammalian artificial chromosomes (MAC) we used site specific recombination mediated by purified Cre recombinase acting on the loxP sequence in PAC vector DNA. A new method was required to purify highly concentrated, virtually 100% intact PAC DNA which could be stored for a long period. Here we show the efficient linking of linearized PACs containing alpha satellite DNA from chromosomes X and 17 with sizes of 125 and 140 kb, respectively, to a 95 kb restriction fragment derived from a 175 kb PAC containing the intact human HPRT gene locus.
Schindelhauer
D
D
MRC, Human Genetics Unit, Western General Hospital, Edinburgh EH4 2XU, UK. dirk@pedgen.med.uni-muenchen.de
Cooke
H J
HJ
D017101:Bacteriophage P1; D002875:Chromosomes; D002876:Chromosomes, Bacterial; D002886:Chromosomes, Human, Pair 17; D004247:DNA / Q000378:metabolism*; D004270:DNA, Circular / Q000378:metabolism; D004276:DNA, Satellite / Q000378:metabolism; D004279:DNA, Viral / Q000737:chemistry; D005821:Genetic Techniques; D006801:Humans; D007041:Hypoxanthine Phosphoribosyltransferase / Q000235:genetics*; D066298:In Vitro Techniques; D019426:Integrases / Q000378:metabolism; D011995:Recombination, Genetic; D014764:Viral Proteins; D014960:X Chromosome
D004270:DNA, Circular; D004276:DNA, Satellite; D004279:DNA, Viral; D014764:Viral Proteins; D004247:DNA; D007041:Hypoxanthine Phosphoribosyltransferase; C045073:Cre recombinase; D019426:Integrases