A novel family of retroviral vectors for the rapid production of complex stable cell lines.
The production of stable cell lines is an important technique in cell biology, and it is often the rate-limiting step in studies involving the characterization of the function of novel genes or gene mutations. To facilitate this process, a novel family of retroviral vectors, the pE vector family, has been generated. The retroviral sequences in the pE vectors have been taken from the Moloney murine leukemia virus (MMLV) vector pMFG, which has been shown to express cDNA inserts more consistently and at higher levels than earlier generations of MMLV vectors. These vectors contain four different internal ribosome entry site-selectable markers, allowing high-efficiency selection of transductants expressing the desired cDNA. The pE vectors have an episomal design to allow long-term production of high-titer virus without the need for subcloning the producer line. Using a strategy of combinatorial infection followed by combinatorial drug selection, we demonstrate that the pE vectors can be used to generate stable, polyclonal cell lines expressing at least three novel cDNAs in less than 2 weeks. The use of these vectors will thus dramatically accelerate the production of complex stable cell lines.
Schaefer
B C
BC
National Jewish Medical and Research Center, Denver, Colorado 80206, USA. schaefer@njc.org
Mitchell
T C
TC
Kappler
J W
JW
Marrack
P
P
D000818:Animals; D018929:Cell Culture Techniques / Q000379:methods*; D002460:Cell Line; D005434:Flow Cytometry; D005822:Genetic Vectors / Q000235:genetics*; D051379:Mice; D008979:Moloney murine leukemia virus / Q000235:genetics* / Q000502:physiology; D010957:Plasmids / Q000235:genetics; D014161:Transduction, Genetic / Q000379:methods*