TAILIEUCHUNG - A new reliable and sensitive nested PCR assay based on the human SRY gene for detection of interspecific chimeras
The present study develops a nested polymerase chain reaction (nPCR) assay to detect human–mouse chimeras. A set of previously validated outer primers, specific to the human SRY gene, was used for conventional one-step PCR, while the inner primers for nPCR were designed. | Turkish Journal of Biology Turk J Biol (2016) 40: 736-746 © TÜBİTAK doi: Research Article A new reliable and sensitive nested PCR assay based on the human SRY gene for detection of interspecific chimeras 1 1 2 1,3 1 1 1 Jie YU , Kaijing LI , Mian HUANG , Liping GUAN , Min ZHANG , Weihua LI , Jianfa HUANG , 1,4 1 1 1 1, Ting LUO , Wencong WANG , Bikun XIAN , Xing LIU , Bing HUANG * 1 State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-Sen University, Guangzhou, . China 2 Guangzhou Zoo, Guangzhou, . China 3 Department of Function Science, Xinxiang Medical College, Henan Xinxiang, . China 4 Guangdong Laboratory Animals Monitoring Institute, Guangzhou, . China Received: Accepted/Published Online: Final Version: Abstract: Although interspecific chimeras can be identified using laborious techniques, more accurate and rapid detectable methods need to be established. The present study develops a nested polymerase chain reaction (nPCR) assay to detect human–mouse chimeras. A set of previously validated outer primers, specific to the human SRY gene, was used for conventional one-step PCR, while the inner primers for nPCR were designed. The specificities of PCR and nPCR assays were examined; both primer sets yielded PCR amplification products from male human epidermis-derived mesenchymal stem cell-like pluripotent cell DNA but no amplification products from negative control DNA. The sensitivity of this nPCR was determined using mixed DNA. Measurable amplification of SRY transcripts was a male human to female mouse DNA ratio of 1:10,000. We then tested the nPCR assay on tissues from female mouse chimeras. The nPCR products were selected randomly for sequencing and positive samples were further analyzed by fluorescence in situ hybridization (FISH) using specific probes for the human SRY gene and by immunofluorescence staining for .
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