Construction and screening of cDNA library of osteosarcoma cells / 南方医科大学学报

ABSTRACT

<p><b>OBJECTIVE</b>To construct a cDNA library of osteosarcoma SAOS-2 cells and screen the proteins interacting with the bone morphogenetic protein-2 (BMP-2) to explore the role of BMP-2 in the development of prostate cancer.</p><p><b>METHODS</b>The total RNA was extracted from SAOS-2 cells, from which poly(A)(+)RNA was purified to generate the cDNA using RT-PCR with primer SMARTIII and primer CDSIIIoligo(dT). The library was constructed and screened by cotransformation of the double-stranded cDNA (dscDNA) and linearized pGADT7-Rec with the bait plasmid to the yeast AH109. The proteins interacting with BMP-2 were screened by AH109 mating with the bait strain Y187, and the interaction was confirmed using far-Western blot analysis.</p><p><b>RESULTS</b>The cDNA library of human osteosarcoma SAOS-2 cells was constructed, which was characterized by high diversity and sufficient capacity. The dscDNA size range was between 250-5000 bp, with a cotransformation efficiency of 4.3 x 10(5) and recombination efficiency of 1.9 x 10(6), and 4.3 x 10(5) clones were screened. The mating efficiency was 32% and 1.0 x 10(6) clones were screened by the bait of BMP-2, and 4 positive clones was obtained and sequenced.</p><p><b>CONCLUSION</b>The diversity and capacity of the cDNA library meet the needs for screening genes related to prostate cancer.</p>