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.