Past Issue

Volume 20, Number 1, Spring 2018, Serial Number: 77 Pages: 98-107

PhiC31-based Site-Specific Transgenesis System for Production of Transgenic Bovine Embryos by Somatic Cell Nuclear Transfer and Intracytoplasmic Sperm Injection


Mohammad Hadi Sekhavati, Ph.D, 1, Sayed Morteza Hosseini, Ph.D, 2, Mojtaba Tahmoorespur, Ph.D, 1, Kamran Ghaedi, Ph.D., 3, 4, Farnoosh Jafarpour, Ph.D., 2, Mehdi Hajian, Ph.D., 2, Kyanoosh Dormiani, Ph.D., 4, Mohammad Hossain Nasr-Esfahani, Ph.D., 2, *,
Department of Animal Science, Ferdowsi University of Mashhad, Mashhad, Iran
Department of Reproductive Biotechnology, Reproductive Biomedicine Research Center, Royan Institute for Biotechnology, ACECR, Isfahan, Iran
Department of Biology, Facualty of Sciences, Uneversity of Isfahan, Isfahan, Iran
Department of Cellular Biotechnology, Cell Science Research Center, Royan Institute for Biotechnology, ACECR, Isfahan, Iran
*Corresponding Address: P.O. BOX: 8165131378 Department of Reproductive Biotechnology Reproductive Biomedicine Research Center Royan Institute for Biotechnology ACECR Isfahan Iran Email:mh.nasr-esfahani@royaninstitute.org

Abstract

Objective

The Streptomyces phage phiC31 integrase offers a sequence-specific method of transgenesis with a robust long-term gene expression. PhiC31 has been successfully developed in a variety of tissues and organs for purpose of in vivo gene therapy. The objective of the present experiment was to evaluate PhiC31-based site-specific transgenesis system for production of transgenic bovine embryos by somatic cell nuclear transfer and intracytoplasmic sperm injection.

Materials and Methods

In this experimental study, the application of phiC31 integrase system was evaluated for generating transgenic bovine embryos by somatic cell nuclear transfer (SCNT) and sperm mediated gene transfer (SMGT) approaches.

Results

PhiC31 integrase mRNA and protein was produced in vitro and their functionality was confirmed. Seven phiC31 recognizable bovine pseudo attachment sites of phage (attP) sites were considered for evaluation of site specific recombination. The accuracy of these sites was validated in phic31 targeted bovine fibroblasts using polymerase chain reaction (PCR) and sequencing. The efficiency and site-specificity of phiC31 integrase system was also confirmed in generated transgenic bovine embryo which successfully obtained using SCNT and SMGT technique.

Conclusion

The results showed that both SMGT and SCNT-derived embryos were enhanced green fluorescent protein (EGFP) positive and phiC31 integrase could recombine the reporter gene in a site specific manner. These results demonstrate that attP site can be used as a proper location to conduct site directed transgenesis in both mammalian cells and embryos in phiC31 integrase system when even combinaed to SCNT and intracytoplasmic sperm injection (ICSI) method.