Expression of a gene encoding a rabbit sperm membrane protein in mammalian cells.
A general mammalian expression vector designated pSV2-EP was reconstructed by inserting an oligonucleotide fragment into pSV2-dhfr. This vector allowed insertion of cDNAs with EcoRI cohesive ends. The pSV2-EP contains a simian virus 40 (SV40) early promoter, origin for DNA replication, SV40 poly-A site, splicing site, an initiator ATG downstream from the promoter and an EcoRI site for the insertion of cDNA fragment screened from lambda gt11 expression libraries. A recombinant plasmid (pS-VRS-1) was constructed by inserting RSD-1, a cDNA encoding a rabbit sperm tail protein, into the EcoRI site of the pSV2-EP vector. Chinese hamster ovarian (CHO) dhfr-negative cells were cotransformed with pSV2-dhfr and pSVRS-1 by the calcium phosphate method. In selective culture medium without thymidine and hypoxanthine, several cell lines were obtained containing mRNA and DNA that hybridized with RSD-1. One of these transformed cell lines stained intensely with anti-rSMP-B antibodies, demonstrating that the RSD-1 was expressed in the transformed CHO cells.
Liu
Q Y
QY
Institute of Basic Medical Sciences, CAMS, Beijing, People's Republic of China.
Wang
L F
LF
Miao
S Y
SY
Zhao
M
M
Zong
S D
SD
Yan
Y C
YC
Koide
S S
SS
D000818:Animals; D000954:Antigens, Surface; D001483:Base Sequence; D016466:CHO Cells; D006224:Cricetinae; D005455:Fluorescent Antibody Technique; D008297:Male; D008565:Membrane Proteins / Q000235:genetics*; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic / Q000235:genetics; D011817:Rabbits; D011994:Recombinant Proteins / Q000096:biosynthesis; D013539:Simian virus 40 / Q000235:genetics; D013094:Spermatozoa / Q000737:chemistry; D014162:Transfection
D000954:Antigens, Surface; D008565:Membrane Proteins; D011994:Recombinant Proteins; C049242:rSMP-B protein, human