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Zhou Z.-W.,Shanghai JiaoTong University | Yan J.-B.,Shanghai JiaoTong University | Yan J.-B.,Key Laboratory of Embryo Molecular Biology | Yan J.-B.,Shanghai Laboratory of Reproduction and Embryo Engineering | And 6 more authors.
Biotechnology Progress | Year: 2011

Recent studies have demonstrated that mitochondrial DNA (mtDNA) haplotype has a significant impact on the efficiency of bovine somatic cell nuclear transfer. Conventional methods for detecting mtDNA variations and haplotypes, such as restriction fragment length polymorphism (RFLP), temporal temperature gradient gel electrophoresis, dHPLC and sequencing, are labor intensive or expensive and have low sensitivity. High-resolution melting (HRM) analysis is a new technique for mutation detection and has the advantages of speed, cost, and accuracy. Here, we describe the genotyping of bovine mtDNA using HRM analysis. DNA samples containing mtDNA were extracted from 75 Holstein cows and subjected to rapid-cycle (<20 min) PCR of small amplicons (<120 bp) using specific primer sets. Capillaries containing the PCR products were then subjected to HRM analysis; data were acquired in 2 min and analyzed using the instrument's software. Five common bovine mtDNA single nucleotide polymorphisms were identified: 9602 G>A, 169 A>G, 166A>G with 173A>G, and 363C>G. These results agree with both sequencing and RFLP analysis. In addition, a very small amount of heteroplasmic variants (<5%) was sufficiently to be distinguished by HRM analysis that would be very useful to differentiate heteroplasmy vs. homoplasmy. HRM analysis thus provides a new approach to genotyping bovine mtDNA sequence variations and has many advantages over other methods, including speed of analysis, cost, and accuracy. We believe this will be a valuable technique for determining the efficiency of nuclear transfer in cloned embryos and for studying maternal effects on nuclear-cytoplasm interactions. © 2011 American Institute of Chemical Engineers.

Li S.,Beijing Jiaotong University | Guo X.,Beijing Jiaotong University | Guo X.,Key Laboratory of Embryo Molecular Biology | Guo X.,Shanghai Laboratory of Reproduction and Embryo Engineering | And 13 more authors.
Biotechnology Letters | Year: 2010

The bovine prolactin vector was injected directly into the mammary glands of mice carrying the human transferrin transgene to investigate its effect on the production of human transferrin in milk. The mean levels of human transferrin in two experimental groups were increased by approx. 60% compared with the control group: 1143 ± 196 ng/ml (experimental group 1; two injections) and 1160 ± 189 ng/ml (experimental group 2; three injections) versus 714 ± 75 ng/ml (control group). These findings suggest the potential utility of the prolactin vector for efficient expression of valuable pharmaceutical proteins in transgenic animal mammary glands. © 2010 Springer Science+Business Media B.V.

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