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Production of Transgenic Goats by Sperm-mediated Exogenous DNA Transfer Method

  • Zhao, Yongju ;
  • Wei, Hong ;
  • Wang, Yong ;
  • Wang, Lingbin ;
  • Yu, Mingju ;
  • Fan, Jingsheng ;
  • Zheng, Shuangyan ;
  • Zhao, Cong
  • Received : 2009.04.07
  • Accepted : 2009.06.22
  • Published : 2010.01.01

Abstract

In this study, the production of transgenic goats using sperm to integrate exogenous DNA and artificial insemination (AI) was carried out and the technical protocols for sperm-mediated gene transfer (SMGT) in the goat were optimized. The standard sperm parameters and the ability to bind foreign genes were assessed to select suitable sperm donor bucks. A total of 134 oestrous does were divided into 4 groups and inseminated using different methods and sperm numbers. The does of Groups I to III were inseminated with fresh semen ($1-2\times10^{7}$ and $10^{6}$ sperm) or frozen-thawed semen ($10^{6}$ sperm), respectively, through conventional intra-cervical AI, and the does of Group IV with frozen-thawed semen ($10^{6}$ sperm) through intrauterine AI. Total genomic DNAs were extracted from ear biopsies of the offspring. The presence of $pEGFP-N_{1}$ DNA was screened by PCR and then by Southern blotting analysis. A total of 76 live kids were produced and 8 kids were tested transgene positive on the basis of agarose gel electrophoresis of the PCR-amplified fragment. Southern blotting analysis of the samples showed 5 positive kids. A transgenic ratio of 10.53% was detected using PCR and 6.58% using Southern blotting. The positive kid rate assayed by PCR and Southern blotting of frozen-thawed goat semen was 3.61% and 9.27% higher than that of untreated semen. The results show that transgenic goats can be produced efficiently by the method of artificial insemination using sperm cells to integrate the exogenous DNA and intrauterine insemination allowed low numbers of DNA-transfected spermatozoa to be used, with satisfactory fertility.

Keywords

Sperm-mediated Gene Transfer;Goat;Sperm Cryopreservation;Artificial Insemination (AI)

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Acknowledgement

Supported by : Shahid Beheshti University