HPRT yeast artificial chromosome transfer into human cells by four methods and an involvement of homologous recombination.
The transfer of a yeast artificial chromosome (YAC) into mouse and human cell lines was effected by four methods, and the efficiency and integrity of the incorporated YAC DNA were compared. A 500 kb YAC containing the human hypoxanthine-guanine phosphoribosyl transferase (HPRT) gene was transferred more efficiently by polyethylene glycol-mediated fusion than by lipofection, electrofusion, or electroporation. Southern blot analysis demonstrated that PEG fusion lines yielded fragments of the size of the original YAC clone, whereas lipofection and electroporation did not. Two of 53 fusion lines showed 6-thioguanine resistance and confirmatory disruption of the HPRT gene in the YAC DNA, suggesting that the YAC DNA was integrated by homologous recombination with the endogenous HPRT gene region.
Wada
M
M
Department of Biochemistry, Kyushu University School of Medicine, Fukuoka, Japan.
Ihara
Y
Y
Tatsuka
M
M
Mitsui
H
H
Kohno
K
K
Kuwano
M
M
Schlessinger
D
D
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D018244:Chromosomes, Artificial, Yeast; D017931:DNA Primers / Q000737:chemistry; D018014:Gene Transfer Techniques; D006801:Humans; D007041:Hypoxanthine Phosphoribosyltransferase / Q000235:genetics*; D066298:In Vitro Techniques; D007739:L Cells; D051379:Mice; D008969:Molecular Sequence Data; D011995:Recombination, Genetic
D017931:DNA Primers; D007041:Hypoxanthine Phosphoribosyltransferase