1. bookVolume 12 (2012): Issue 3 (July 2012)
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Journal
eISSN
2300-8733
ISSN
1642-3402
First Published
25 Nov 2011
Publication timeframe
4 times per year
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English
Open Access

Determination of the Absolute Number of Transgene Copies in CMVFUT Transgenic Pigs

Published Online: 24 Jul 2012
Volume & Issue: Volume 12 (2012) - Issue 3 (July 2012)
Page range: 349 - 356
Journal Details
License
Format
Journal
eISSN
2300-8733
ISSN
1642-3402
First Published
25 Nov 2011
Publication timeframe
4 times per year
Languages
English
Determination of the Absolute Number of Transgene Copies in CMVFUT Transgenic Pigs

The aim of this research was to determine the number of transgene copies in the DNA of transgenic pigs. The copy number of the transgene was analysed in the transgenic animals with introduced pCMVFUT genetic construct containing a coding sequence of human H transferase under a control of CMV promoter. The copy number of the transgene that had integrated with the genome of the transgenic animals was analysed by qPCR with SYBR Green dye, which enabled nonspecific double-stranded DNA detection. CMVFT-2F and CMVFT-2R primers were used to amplify a 149 bp fragment of DNA. Forward primer had a sequence complementary to a promoter sequence and reverse primer to a coding sequence of H transferase. The copy number of the transgene in the examined samples was established by plotting the CT values obtained on a standard curve, which had been set by the usage of the CT values for the successive standard dilutions with known copy number (1.438-1.431 copies). As a standard we used pCMVFut genetic construct hydrolyzed with Not I restriction enzyme to a linear form. The real-time PCR results helped to establish the range of 3 - 4 as the number of the transgene copies that had integrated to the swine genome.

Keywords

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