Incorporation and expression of a neo gene after transfer to caprine hematopoietic cells using a modified retrovirus.
The ability of the N2 retrovirus to introduce the selectable neo gene into (transform) caprine hematopoietic cells (CHC) was evaluated. Helper-free amphotropic retrovirus producing cells (RPC) were plated at approximately 1.0 x 10(5) cells per 25 cm2 tissue culture flask, cultured to 80% confluence and irradiated (1,500 rads) prior to CHC incubation. The CHC collected from three donor goats were washed and cultured for 24 h in either the RPC or control flasks. Cells were cultured in Dulbecco's Modified Eagles Medium containing 10% fetal calf serum (FCS) (DMEM) and 4 micrograms polybrene/ml. After 48 h of culture in fresh DMEM, cells were recovered and suspended in Iscove's DMEM supplemented with .3% gar, 12% FCS and 400 micrograms geneticin/ml (G418; neomycin) and transferred to 35-mm petri dishes (7.5 x 10(5) cells) for selection of G418 resistant cells. After 17 d of culture, plates were evaluated for total number of colony forming units (CFU, greater than 10 cells). Total number of CFU was greater (P less than .01) in treatment samples (means = 175, SEM = 70) than in control cultures (mean = 0, SEM = 0). The N2 retrovirus appears to be an effective vector for the transformation of CHC and may provide a means to introduce gene(s) into cells of domestic animals.
Casey
D C
DC
Dept. of Anim. Sci., Louisiana State University, Baton Rouge.
White
K L
KL
D000818:Animals; D002460:Cell Line; D004352:Drug Resistance, Microbial / Q000235:genetics; D005260:Female; D015870:Gene Expression; D005822:Genetic Vectors; D006041:Goats / Q000235:genetics*; D006412:Hematopoietic Stem Cells / Q000502:physiology*; D012190:Retroviridae / Q000235:genetics; D014162:Transfection