Abstract:
: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.
journal_name
Proc Natl Acad Sci U S Aauthors
Kitamura T,Onishi M,Kinoshita S,Shibuya A,Miyajima A,Nolan GPdoi
10.1073/pnas.92.20.9146subject
Has Abstractpub_date
1995-09-26 00:00:00pages
9146-50issue
20eissn
0027-8424issn
1091-6490journal_volume
92pub_type
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