• Reproductive and Developmental Biology
• /
• v.30 no.3
• /
• pp.189-194
• /
• 2006

Sperm-mediated gene transfer(SMGT) can be used to transfer exogenous DNA into the oocyte at fertilization. The main objective of this study was to assess efficiency of transferring mitochondrial DNA(mtDNA) fragment into boar spermatozoa in either presence or absence of liposome and quality of transfected spermatozoa. The mtDNA of chicken liver was isolated and purified by phenol and alkaline lysis extraction, and it was inserted to plasmid. The genome of transfected spermatozoa treated with DNase I was purified by alkaline lysis, and then amplified by the PCR analysis. After electrophoresis, DNA quantitation of each well was calculated by comparison of the band intensity with standard. As a result, exogenous DNA was composed of mtDNA fragment(1.2 kb) and plasmid(2.7 kb). On the other hand, efficiency of transfection by liposome($9.0{\pm}0.34ng/{\mu}l$) in SMGT was higher than that by DNA solution($6.9{\pm}0.53ng/{\mu}l$). However, there was no significant difference. Transfering exogenous DNA into spermatozoa was completed within 90 min of incubation. In another experiment, there were significant (p<0.05) differences between transfected spermatozoa using both DNA solution and DNA/liposome completes with unheated spermatozoa for viability ($70.8{\pm}1.80$ and $68.0{\pm}2.16%$ vs. $83.3{\pm}1.69%$, respectively) and motility($78.7{\pm}1.59$ and $79.3{\pm}2.14%$ vs. $86.7{\pm}1.59%$, respectively). This study indicates that exogenous mtDNA can be efficiently transferred into boar spermatozoa regardless of the presence of liposome, and transfected spermatozoa can also use insemination and in vitro fertilization to generate transgenic pig.

• Asian-Australasian Journal of Animal Sciences
• /
• v.17 no.7
• /
• pp.885-891
• /
• 2004

To evaluate the feasibility of using sperm-mediated gene transfer (SMGT) for carrying foreign gene into chicken oocyte, a reporter gene, CX-EGFP, was used in this study. The reporter gene was first mixed with liposome or liposome-like compound and the mixtures were further combined with ejaculated cock spermatozoa. The spermatozoa treated with liposome and CX-EGFP mixture was subsequently coincubated with DNaseI to remove the extra DNA which insured the authenticity of positive signals. The treated sperms were then subjected to transgene (reporter gene) existence analysis and artificial insemination of laying hens. Obtained results indicated that the spermatozoa were able to take-in the foreign DNA; which was confirmed by polymerase chain reaction and Southern blot analysis. In the following experiment, fresh ejaculated sperms were mixed with CX-EGFP-liposome or CX-EGFP-liposome-like complex then used for artificial insemination of each of six laying hens. Eggs laid between day-3 and day-7 post insemination were collected. Newly hatched chicks, two out of 53 from CX-EGFP/liposome treated group and two out of 21 from CXEGFP/liposome-like treated group, were proven to be transgenic. This study suggests that SMGT is a powerful method for generating transgenic chickens.

• Asian-Australasian Journal of Animal Sciences
• /
• v.23 no.1
• /
• pp.33-40
• /
• 2010

In this study, the production of transgenic goats using sperm to integrate exogenous DNA and artificial insemination (AI) was carried out and the technical protocols for sperm-mediated gene transfer (SMGT) in the goat were optimized. The standard sperm parameters and the ability to bind foreign genes were assessed to select suitable sperm donor bucks. A total of 134 oestrous does were divided into 4 groups and inseminated using different methods and sperm numbers. The does of Groups I to III were inseminated with fresh semen ($1-2\times10^{7}$ and $10^{6}$ sperm) or frozen-thawed semen ($10^{6}$ sperm), respectively, through conventional intra-cervical AI, and the does of Group IV with frozen-thawed semen ($10^{6}$ sperm) through intrauterine AI. Total genomic DNAs were extracted from ear biopsies of the offspring. The presence of $pEGFP-N_{1}$ DNA was screened by PCR and then by Southern blotting analysis. A total of 76 live kids were produced and 8 kids were tested transgene positive on the basis of agarose gel electrophoresis of the PCR-amplified fragment. Southern blotting analysis of the samples showed 5 positive kids. A transgenic ratio of 10.53% was detected using PCR and 6.58% using Southern blotting. The positive kid rate assayed by PCR and Southern blotting of frozen-thawed goat semen was 3.61% and 9.27% higher than that of untreated semen. The results show that transgenic goats can be produced efficiently by the method of artificial insemination using sperm cells to integrate the exogenous DNA and intrauterine insemination allowed low numbers of DNA-transfected spermatozoa to be used, with satisfactory fertility.