Sperm status and DNA dose play key roles in sperm/ICSI‐mediated gene transfer in caprine

Abstract

In relation to the growing recent interest in the establishment of sperm-mediated gene transfer (SMGT) technology as a convenient and effective method for the simple production of transgenic animals, in this study the possibility of using SMGT to produce transgenic caprine embryos was investigated for the first time. Buck sperm were directly incubated with different concentrations (0-500 ng) of pcDNA/his/Lac-Z plasmid and used for IVF or ICSI. Sperm used for ICSI were categorized into motile or live-immotile group before being injected into oocytes. In a separate experiment, dead sperm prepared by repeated freezing/thawing were used for DNA-incubation before ICSI. Sham injection was carried out by intracytoplasmic injection of approximately the same volume of media containing different doses of DNA using an ICSI needle. Transgene expression and transmission were detected by X-Gal staining and PCR analysis of developed embryos, respectively. A reasonable blastocyst rate was observed in all the groups. Only embryos in the sham group were negative for transgene transmission. Transgene expression was completely dependent on the delivery technique and status of sperm, and was only observed in the live-immotile and dead ICSI groups. The results of this study showed that the technique (IVF vs. ICSI vs. sham injection), sperm status (motile vs. live-immotile vs. dead) and to some extent DNA concentration affect embryo development, transgene transmission and expression.

Development of an Optimized Zona-Free Method of Somatic Cell Nuclear Transfer in the Goat

“…Matured COCs in different IVM treatments (Fig. 1C) were then subjected to in vitro fertilization as described by Shadanloo et al (2010) before being cultured in an optimized formulation of synthetic oviduct fluid (mSOF). The mSOF was a serum-free continuous formulation of Tervit et al, (1972), which was supplemented with essential and nonessential amino acids (2% and 1%, respectively), EGF (100 ng/mL), IGF (100 ng/mL), and an optimized serum substitute [B-27, 2% (v/v)] which enabled high in vitro development of goat embryos (unpublished data) ( Fig.…”

Exposure to DNA is insufficient for in vitro transgenesis of live bovine sperm and embryos

“…K-SIPV-200-5; Cook Medical, Brisbane, QLD, Australia) drops and immotile sperm injected into matured cumulus-free oocytes with an extruded first polar body. Oocytes were chemically activated (Shadanloo et al 2010) and allowed to extrude the second polar body for 3 h, before incubation in 2 mM 6-dimethyl aminopurine for 4 h (Shadanloo et al 2010). Immotile sperm without DNA incubation underwent the same chemical activation and served as sham control.…”

“…This route circumvents membrane fusion and therefore does not rely on sperm internalising exogenous DNA. It results in conclusive DNA uptake and reproducible reporter gene expression in transgenic embryos and animals (Perry et al 1999, Chan et al 2000, Lai et al 2001, Pereyra-Bonnet et al 2008, Garcia-Vazquez et al 2009, Wu et al 2009, Shadanloo et al 2010.…”

Improved Bovine ICSI Outcomes by Sperm Selected after Combined Heparin-Glutathione Treatment

“…ICSI was performed using an injection pipette with an inner diameter at the tip of 8 lm for injection of control sperm and ‡ 12 lm for injection of decondensed sperm. ICSI was carried out as described previously (Shadanloo et al, 2010). Chemical activation of injected oocytes was performed out according to Nasr-Esfahani et al (2008) with minor modifications.…”