Study On Sperm-mediated Gene Transfer In Pig With Nano-PAMAM-D

To optimize production of transgenic pig by sperm-mediated gene transfer, the content of present study contains:(1) The present study was designed to examine the effects of treatment with Nano-PAMAM-D on exogenous DNA uptake of boar spermatozoa and gene expression in in-vitro fertilized embryos. Exogenous DNA treated with Nano-PAMAM-D was incubated with boar spermatozoa, the exogenous DNA uptake was detected by in situ hybridization, and exogenous gene expression was examined by fluorescence microscope and PCR analysis. The positive rate of sperm treated with Nano-PAMAM-D and DNA according to0.5μg linear plasmid plus PAMAM-D when the ratio of20:1was the highest group (P<0.05).The positive rates of were significantly increased by incubation90or120min compared to incubation30or60min(P<0.05). The complexes formed between exogenous DNA and Nano-PAMAM-D were internalized (23.93%±2.60%vs7.25%±2.04%,13.25%±2.15%, P<0.05) on boar spermatozoa at a higher efficiency compared to methods using DNA alone or lipofectin. This study suggested that boar spermatozoa using Nano-PAMAM-D as transfectant coμLd be fertilized with oocytes in vitro and that the resμLting gene of green fluorescent protein was detected in fertilized embryos by genomic PCR analysis. (2) We investigated whether the expression of transgenic DNA in SMGT-ICSI embryos is improved by PAMAM-D-mediated DNA transfer. EGFP-expressing embryos were obtained with a higher efficiency using the SMGT-ICSI technique in combination with PAMAM-D compared to control group.(25.83%±2.31%vs17.5%±1.80%; P<0.05); It is efficient for the development of SMGT-ICSI embryos and EGFP-expression using1.0kV/cm,30μs,1DC as electronic activation parameter and2Io+DMAP as chemical activation protocol.(3)The linearized plasmid DNA contained phytase gene was mixed with the new collected semen removed the fluid, then incubated at17℃for90min.6recipient sows were insemined by artificial insemination. The4pregnant sows generated38piglets, and6positive transgenic pig was detected by PCR.In conclusion, Nano-PAMAM-D vector coμLd be used to efficiently introduce a exogenous gene into embryo via spermatozoa. EGFP-expression is higher efficiency using the SMGT-ICSI technique in combination with PAMAM-D.The resμLts also demonstrated that the phytase gene had transferred into the positive piglets, and the feasibility of sperm-mediated gene transfer to produce transgenic porcine.