Transgene transmission in chickens by sperm-mediated gene transfer after seminal plasma removal and exogenous DNA treated with dimethylsulfoxide or N,N-dimethylacetamide

Abstract

Transgenic animals have been successfully produced by mass gene transfer techniques such as sperm-mediated gene transfer (SMGT). The aim of this work was to demonstrate transgene transmission by SMGT in chickens using dimethylsulfoxide (DMSO) or N,N-dimethylacetamide (DMAc) as transfectants after seminal plasma removal to prevent DNase activity. Sperm samples were prepared by repetitive washes, and after each wash sperm motility, seminal plasma proteins, exogenous DNA integrity and its uptake by spermatozoa were evaluated. Laying hens were inseminated using spermatozoa transfected with pEGFP-N1 vector in the presence of DMSO or DMAc. Transgene transmission in newborn chicks was evaluated by in vivo enhanced green fluorescent protein (EGFP) expression, RT-PCR and PCR analysis. DNA internalization was limited to sperm samples washed twice. The presence of DMSO or DMAc during transfection had no effect on fertilization or hatching rates. PCR analysis detected the presence of EGFP DNA in 38% of newborn chicks from the DMSO group and 19% from the DMAc group. EGFP mRNA was detected in 21% of newborn chicks from the DMSO group, as against 8.5% from the DMAc group. However, in vivo expression of EGFP was only observed in a single animal from the DMSO group. Our data revealed that the plasmid DNA-DMSO combination coupled with sperm washes can be an efficient method for transfection in chickens.

Testis-mediated gene transfer in mice: comparison of transfection reagents regarding transgene transmission and testicular damage

“…On the other hand, intracellular cryoprotectants such as DMSO can improve DNA uptake by sperm cells as previously demonstrated in rabbits, mice and chicken Shen et al, 2006, Collares et a., 2011. Therefore other intracellular cryoprotectants such as N, N-dimetylacetamide (DMA) that have been used recently in boar sperm cryopresenvation (Bianchi et al, 2008) could also be used to increase the uptake of exogenous DNA by spermatozoa as demonstrated for chickens (Collares et al, 2011).…”

“…Therefore other intracellular cryoprotectants such as N, N-dimetylacetamide (DMA) that have been used recently in boar sperm cryopresenvation (Bianchi et al, 2008) could also be used to increase the uptake of exogenous DNA by spermatozoa as demonstrated for chickens (Collares et al, 2011). …”

RNA interference-based technology: what role in animal agriculture?

“…In this method the transgene containing the shRNA is attached or inserted into the sperm head and then used to fertilise eggs (Moreira et al 2007). DNA can be introduce to the sperm head in several ways including transfection (Collares et al 2011), attachment by antibodies (Chang et al 2002) and disruption by repeated freeze-thaw cycles or exposure to detergents (Perry et al 2001). Random insertion of the shRNA construct into the genome can lead to problems including overloading of the RNAi machinery (Grimm et al 2006), complete elimination of a required gene product, or epigenetic silencing of the construct.…”

Visible Diode Laser Enhancement of Exotic DNA Uptake by Fowl Sperm

“…El-Gendy et al [12] successfully developed the F 1 transgenic chickens by SMGT and using lipofectin as a transfection reagent. Also, Collares et al [36] reported successful transmission of an exogenous DNA to chickens by SMGT after removing seminal plasma and treating the exogenous DNA with dimethylsulfoxide or N,Ndimethylacetamide. Lubart et al [37] indicated that there is an accelerated exchange and uptake of Ca 2+ between the medium and the bovine sperm cells during irradiation with 6-8 J/cm 2 He-Ne (633 nm).…”