Evaluating bovine sperm transfection using a high-performance polymer reagent and assessing the fertilizing capacity of transfected spermatozoa using an in vitro fertilization technique

Ali Jafarnejad,Mohammad Zandi,Mehdi Aminafshar,Naser Emamjomeh Kashan,Mohammad Reza Sanjabi

doi:10.5194/aab-61-351-2018

Abstract

Abstract. Sperm-mediated gene transfer (SMGT) has been considered as an innovative device for transgenesis on a mass scale by taking advantage of live spermatozoa to transfer exogenous DNA. However, the fertilizing ability of transfected sperm cells and the poor reproducibility of this method are still matters of controversy. Hence, the current study was conducted to evaluate transfecting the enhanced green fluorescent protein (EGFP) as the source of exogenous DNA into bovine spermatozoa using a high-performance polymer reagent as well as assessing the fertilizing capacity of transfected sperm cells by in vitro fertilization (IVF). In the first experiment, three different concentrations of rhodamine-labeled DNA and high-performance polymer transfection reagent, X-tremeGENE HP, were used to transfect bovine spermatozoa. In the second experiment, IVF and fluorescence microscopy methods were utilized to assess the fertilizing capacity of sperm cells carrying exogenous DNA when X-tremeGENE HP was used either alone or with dimethyl sulfoxide (DMSO) treatment. Findings revealed that at 1 µL X-tremeGENE HP and 1 µg of DNA concentration, approximately one-third of total spermatozoa were transfected. However, following IVF and fluorescence microscopy, no EGFP expression was detected in zygotes and morula-stage embryos. Results of this study showed that, although X-tremeGENE HP could transfer EGFP to bovine spermatozoa, transfected sperm cells were unable to transfer foreign DNA to matured bovine oocytes. Under our experimental conditions, we hypothesized that the absence of the EGFP fluorescence signal in embryos could be due to the detrimental effects of transfection treatments on sperm cells' fertility performance as well as incompetency of IVF to produce transgenic embryos using transfected sperm cells.

Highlights

Over the past three decades, several procedures including pronuclear micro-injection (PNM), somatic cell nuclear transfer (SCNT) and viral infection (VI) have been applied and developed to introduce foreign genes into different recipient cells (Eghbalsaied et al, 2009)
Findings of the present study showed that among three different amounts of DNA and reagents, 1 μL X-tremeGENE HP and 1 μg of DNA were the best and effective concentrations for transfection of bovine spermatozoa
Following in vitro fertilization (IVF), in vitro cultured embryos were monitored by fluorescence microscopy; no sign of the presence of transgene was observed in zygotes, and all morula were enhanced green fluorescent protein (EGFP) negative

Summary

Introduction

Over the past three decades, several procedures including pronuclear micro-injection (PNM), somatic cell nuclear transfer (SCNT) and viral infection (VI) have been applied and developed to introduce foreign genes into different recipient cells (Eghbalsaied et al, 2009). These techniques had certain drawbacks including low efficiency, random integration sites, high mosaic rate, complexity and inability to transfect large DNA fragments (Eghbalsaied et al, 2009). Factors including the kind and the amount of DNA constructs (Canovas et al, 2010), as well as the gene transfer technique (Alderson et al, 2006), have been reported as some effective factors which can deeply affect the efficiency rate of transfection

Methods

Results

Discussion

Conclusion