Avian retrovirus integrase-enhanced transgene integration into mammalian cell DNA in vivo.
Systems for introducing DNA genes-of-interest into mammalian cellular genomes have ranged from the use of different physical techniques to viruses including retroviruses. We have developed a microinjection method for an efficient and permanent integration of a DNA transgene into the cell genome by use of the retrovirus integrase. A 3.0-kb linear DNA fragment containing an internal herpes simplex virus thymidine kinase gene (tk) with flanking avian retrovirus U5 and U3 terminal attachment sites (U5-pgk/tk-U3) recognized by the integrase was constructed. The other donor, a 3.3-kb linear DNA fragment containing the same gene (pgk/tk) flanked by ApaL1 restriction sites not recognized by integrase, was also produced. After assembly of integrase-transgene complexes on ice, the complexes were microinjected into the nucleus of human fibroblast cells (143Btk) containing a defective thymidine kinase. The number of hypoxanthine/aminopterin/thymidine (HAT)-resistant colonies produced upon microinjection of either naked DNA or the independently assembled integrase-transgene complexes were determined. Our data suggests that enhanced integration of U5-pgk/tk-U3 required the DNA attachment sites and co-delivery of integrase. The data was consistent with a direct role for both of these elements in producing an approximate 4-fold increase in the number of HAT-resistant colonies observed over microinjection of just naked U5-pgk/tk-U3 (P < 0.0001).
Yao
Aqing
A
University of Texas Medical Branch, Galveston, TX, USA.
Chiu
Roger
R
Vora
Ajaykumar
A
Brown
David B
DB
Grandgenett
Duane
D
Davis
Brian R
BR
D000818:Animals; D002460:Cell Line; D004247:DNA / Q000008:administration & dosage*; D005347:Fibroblasts / Q000502:physiology; D018014:Gene Transfer Techniques; D006801:Humans; D019426:Integrases / Q000235:genetics* / Q000378:metabolism; D008322:Mammals; D008845:Microinjections / Q000379:methods*; D010957:Plasmids / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012191:Retroviridae Proteins / Q000235:genetics* / Q000378:metabolism; D014161:Transduction, Genetic / Q000379:methods*; D019076:Transgenes / Q000235:genetics*
D011993:Recombinant Fusion Proteins; D012191:Retroviridae Proteins; C040520:avian retrovirus proteins; D004247:DNA; D019426:Integrases