Targeted gene modification in mouse ES cells using integrase-defective lentiviral vectors.
Lentiviral vectors efficiently integrate into the host genome of both dividing and nondividing cells, and so they have been used for stable transgene expression in biological and biomedical studies. However, recent studies have highlighted the risk of insertional mutagenesis and subsequent oncogenesis. Here, we used an integrase-defective lentiviral (IDLV) vector to decrease the chance of random integration and examined the feasibility of lentiviral vector-mediated gene targeting into murine embryonic stem (ES) cells. After transduction with wild-type lentiviral vectors, none of the 512 G418 resistant clones were found to be homologous recombinant clones. Although the transduction efficiency was lower with the IDLV vectors (5.9% of wild-type), successful homologous recombination was observed in nine out of the 941 G418 resistant clones (0.83 +/- 1.32%). Pluripotency of the homologous recombinant ES cells was confirmed by the production of chimeric mice and subsequent germ line transmission. Because lentiviral vectors can efficiently transduce a variety of stem cell types, our strategy has potential relevance for secure gene-manipulation in therapeutic applications.
Okada
Yuka
Y
Research Institute for Microbial Diseases, Osaka University, Suita, Osaka, Japan.
Ueshin
Yuko
Y
Hasuwa
Hidetoshi
H
Takumi
Kazuhiro
K
Okabe
Masaru
M
Ikawa
Masahito
M
D000818:Animals; D000831:Animals, Newborn; D001757:Blastomeres / Q000166:cytology / Q000378:metabolism; D015139:Blotting, Southern; D002135:Calcium-Binding Proteins / Q000235:genetics; D003673:Defective Viruses / Q000235:genetics; D053595:Embryonic Stem Cells / Q000166:cytology / Q000378:metabolism* / Q000637:transplantation; D005260:Female; D018390:Gene Targeting / Q000379:methods*; D005822:Genetic Vectors / Q000235:genetics*; D019426:Integrases / Q000235:genetics*; D016086:Lentivirus / Q000235:genetics; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D008813:Mice, Inbred ICR; D008957:Models, Genetic; D018832:Molecular Chaperones / Q000235:genetics; D016254:Mutagenesis, Insertional; D039904:Pluripotent Stem Cells / Q000166:cytology / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011247:Pregnancy; D033581:Stem Cell Transplantation; D014162:Transfection / Q000379:methods
D002135:Calcium-Binding Proteins; D018832:Molecular Chaperones; C087341:calmegin; D019426:Integrases