Applications of retrovirus-mediated expression cloning.
We have recently established a novel expression cloning system using retroviral vectors. The system is based on a high-efficiency packaging cell line, BOSC23, and a simplified retroviral vector, pBabeX, carrying no selection marker. cDNA libraries, constructed in the pBabeX vector, are transiently transfected into BOSC23 cells. The supernatant contains more than 3X10(6)/mL, which would cover large complexities of cDNA libraries. The retrovirus stock gave 100% infection efficiency in NIH3T3 cells and 5-40% infection efficiency in various hematopoietic cell lines. In contrast to the conventional expression cloning system, in which it is necessary to transfect cDNA libraries transiently into particular cell types such as COS cells, retrovirus-mediated expression cloning allows us to transduce cDNAs into a wide variety of cell types. This method therefore makes it possible to select cells expressing a cDNA of interest by various functional assays. When combined with polymerase chain reaction (PCR)-driven random mutagenesis, this system is also useful in searching for mutations of various molecules that will result in alterations of their functions.
Onishi
M
M
DNAX Research Institute of Molecular and Cellular Biology, Palo Alto, CA 94304, USA.
Kinoshita
S
S
Morikawa
Y
Y
Shibuya
A
A
Phillips
J
J
Lanier
L L
LL
Gorman
D M
DM
Nolan
G P
GP
Miyajima
A
A
Kitamura
T
T
D016475:3T3 Cells; D000818:Animals; D000954:Antigens, Surface / Q000096:biosynthesis / Q000235:genetics; D002460:Cell Line; D003001:Cloning, Molecular / Q000379:methods*; D004252:DNA Mutational Analysis / Q000379:methods; D018076:DNA, Complementary / Q000235:genetics; D004274:DNA, Recombinant / Q000235:genetics; D015967:Gene Expression Regulation, Viral; D015723:Gene Library; D005822:Genetic Vectors; D006412:Hematopoietic Stem Cells; D051379:Mice; D016296:Mutagenesis; D010714:Phosphatidylethanolamines; D016133:Polymerase Chain Reaction; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics; D012190:Retroviridae / Q000235:genetics*; D016662:Virus Integration
D000954:Antigens, Surface; D018076:DNA, Complementary; D004274:DNA, Recombinant; D010714:Phosphatidylethanolamines; D011993:Recombinant Fusion Proteins; C020888:1,2-dielaidoylphosphatidylethanolamine