Cloning and expression of cynomolgus monkey and baboon zona pellucida proteins.
Partial clones for the three cynomolgus monkey (Macaca fasicularis) zona pellucida genes (cmZPA, cmZPB, and cmZPC) have previously been isolated. These partial clones contained the sequences for the C-terminal portion of each rcmZP protein. To obtain full-length clones for each cmZP, a fresh cynomolgus monkey ovarian cDNA library was constructed. PCR methodology was employed to speed the isolation of full-length clones for each cmZP cDNA. The 3' primers were designed based on sequence information from the previously identified clones; the 5' primers were designed using the human ZP sequences. The PCR technique yielded full-length clones of cmZPA and cmZPC, but not of cmZPB. Therefore, a genomic clone of cmZPB was isolated and the sequence determined. The exon/intron structure is nearly identical to the human ZPB exon/intron structure. New PCR primers were designed based on the cynomolgus monkey ZPB genomic sequence, and a full-length cmZPB cDNA was obtained. The same primers that were used to generate the cmZPB were also used to generate a baboon (Papio cynocephalus) ZPB (bZPB) cDNA. As was done previously for the human zona pellucida (hZP) cDNAs, the cmZP, and bZPB cDNAs were transferred to shuttle vectors for transfection into Chinese Hamster Ovary (CHO) cells. Stable cell lines for producing each ZP protein were isolated. Each cell line secreted the desired recombinant zona pellucida (rZP) protein into the culture medium, and each protein was purified using an established protocol. In terms of size and purity, the purified recombinant cmZP (rcmZP) and rbZPB proteins resemble the rhZP proteins.
Harris
Jeffrey D
JD
Zonagen, Inc., The Woodlands, Texas 77380, USA. jdh@zonagen.com
Piersen
Colleen E
CE
D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers; D018076:DNA, Complementary / Q000235:genetics / Q000302:isolation & purification; D004527:Egg Proteins / Q000235:genetics*; D005091:Exons; D005260:Female; D005786:Gene Expression Regulation; D006801:Humans; D007438:Introns; D008252:Macaca fascicularis; D008562:Membrane Glycoproteins / Q000235:genetics*; D010215:Papio; D016133:Polymerase Chain Reaction; D011956:Receptors, Cell Surface; D015183:Restriction Mapping; D015044:Zona Pellucida / Q000502:physiology*; D000072216:Zona Pellucida Glycoproteins
D017931:DNA Primers; D018076:DNA, Complementary; D004527:Egg Proteins; D008562:Membrane Glycoproteins; D011956:Receptors, Cell Surface; D000072216:Zona Pellucida Glycoproteins