Efficient screening of retroviral cDNA expression libraries.
Expression cloning of cDNAs was first described a decade ago and was based on transient expression of cDNA libraries in COS cells. In contrast to transient transfection of plasmids, retroviral gene transfer delivers genes stably into a wide range of target cells. We utilize a simple packaging system for production of high-titer retrovirus stock from cDNA libraries to establish a cDNA expression cloning system. In two model experiments, murine interleukin (IL)-3-dependent Ba/F3 cells were infected with libraries of retrovirally expressed cDNA derived from human T-cell mRNA or human IL-3-dependent TF-1 cell line mRNA. These infected Ba/F3 cells were selected for the expression of CD2 by flow cytometry or for the alpha subunit of the human IL-3 receptor (hIL-3R alpha) by factor-dependent growth. CD2 (frequency, 1 in 10(4)) and hIL-3R alpha (frequency, 1 in 1.5 x 10(5)) cDNAs were readily detected in small-scale experiments, indicating this retroviral expression cloning system is efficient enough to clone low-abundance cDNAs by their expression or function.
Kitamura
T
T
Department of Cell Biology, DNAX Research Institute of Molecular and Cell Biology, Palo Alto, CA 94304, USA.
Onishi
M
M
Kinoshita
S
S
Shibuya
A
A
Miyajima
A
A
Nolan
G P
GP
D016475:3T3 Cells; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D002522:Chlorocebus aethiops; D003001:Cloning, Molecular; D018076:DNA, Complementary; D016208:Databases, Factual; D004526:Efficiency; D005822:Genetic Vectors; D006801:Humans; D007377:Interleukin-3 / Q000494:pharmacology; D007668:Kidney; D051379:Mice; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D012333:RNA, Messenger / Q000378:metabolism; D016185:Receptors, Interleukin-3 / Q000096:biosynthesis*; D011994:Recombinant Proteins / Q000096:biosynthesis*; D015183:Restriction Mapping; D012190:Retroviridae; D013601:T-Lymphocytes / Q000378:metabolism; D014162:Transfection
D018076:DNA, Complementary; D007377:Interleukin-3; D009838:Oligodeoxyribonucleotides; D012333:RNA, Messenger; D016185:Receptors, Interleukin-3; D011994:Recombinant Proteins