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

OBJECTIVES: 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. CONCLUSIONS: 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.

Chances to Have A Boy after Gender Selection by Pre-Implantation Genetic Screening Are Reduced in Couples with only Girls and without A Boy Sired by The Male Partner.

BACKGROUND: Gender selection and family planning have their roots in human history. Despite great interest in these fields, very few scientific propositions exist which could explain why some family do not attain the desired sex. Therefore, the aim of this study was to evaluate whether sex of previous child or children could affect the outcomes of pre-implantation genetic screening (PGS). MATERIALS AND METHODS: This historical cohort study including 218 PGS cases referring to Isfahan Fertility and Infertility Center (IFIC). Couples were grouped as those who their male child passed away or her husbands' has a son(s) from their previous marriage (n=70) and couples who just have daughter (n=148). Male normal blastocysts were transferred for both groups. The outcomes of PGS including pregnancy, implantation and abortion rates, along with possible confounding factors were compared between the two groups. RESULTS: Significant differences in pregnancy, implantation and abortion rates were observed between couples whose their male partner had/has one boy (n=70) compared to those who have just girl(s) (n=148) despite similar number and quality of male normal blastocyst transferred in the two groups. Confounding factors were also considered. CONCLUSION: The Ybearing spermatozoa in male partners with no history of previous boy have lower ability to support a normal development to term, compared to male partners with previous history of boy requesting family balancing.