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KMID : 0900919930170010043
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Korean journal of Animal Reproduction
1993 Volume.17 No. 1 p.43 ~ p.48
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Molecular Cloning of H-Y Antigen Gene III. Construction of Mouse Testis cDNA Library and Screening of H-Y Ag Gene
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Kim Jong-Bae
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Abstract
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These experiments were carried out to construct mouse testis cDNA library and to to seen H-Y Ag gene. Mouse testis was obtained from BALB/c inbreed mouse that was after-born 1 week. Isolation of mouse testis total RNA was carried out by guanidum/cesium choloride, poly(A+) mRNAs were purified by oligo d(T)-cellulose chromatography method. To investigate protein synthesis activity, in-vitro translation carried out by total RNA and poly(A+) mRNA. The products of in-vitro translation were identified in 12.5% PAGE. Single strand DNA and double strand DNA were synthesized from poly(A+) mRNA and purified using phenol/chloroform/isoamylalcohol. Synthesized cDNA was combined with cohesive Eco RI polylinker, its recombination efficiencies were identified by X-gal and IPTG. In the cDNA library, 1times107 phagemids were screened with 32P labelled probe. Hybridization were carried on 65^{circ}C for 16~20hours. And 1times106 phagemids were screened with rabbit-anti-H-Y. In former, select 5 positive clones, and later, 1 positive clone. Its southern blot analysis showed various size of insert cDNA from 0.7kb to 3kb.
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