• Reproductive and Developmental Biology
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• v.32 no.2
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• pp.81-87
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• 2008

Production of transgenic animals for studying specific gene has been limited due to a low efficiency, lack of skilled researchers and the need for expensive equipment. Currently, the boar spermatozoa as a vector to deliver exogenous DNA into the oocyte were used to improve the efficiency of transfection rate. In this study, we revealed that the optimal conditions for DNA uptake in spermatozoa by liposome were to 90 min of incubation, $17^{\circ}C$, $10^5$ spermatozoa, 4 ng/ml of exogenous DNA and 0.5% (v/v) liposome, without damage to fertility. In addition, the developmental rate to the blastocyst stage of embryo in control group was significantly higher than those embryos with exogenous DNA and liposome, whereas there were no significant differences in embryo development between the liposome and type of DNA. The transfection rates of embryo using treated spermatozoa with both liposome and circular DNA were higher than those using linear DNA. These findings raise the possibility thattreated spermatozoa with liposome/DNA complexes could be used in in vitro fertilization, and the exogenous DNA transferred into the oocytes. Taken together, we demonstrated that liposome a vector for the uptake of exogenous DNA in boar spermatozoa could improve the efficiency of sperm-mediated gene transfer in creating transgenic pig and the other domestic transgenic animals.

• Asian-Australasian Journal of Animal Sciences
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• v.15 no.10
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• pp.1412-1421
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• 2002

To examine the feasibility of using a sperm vector system for gene transfer, we have investigated the binding and the uptaking of foreign DNA into the sperm nucleus by PCR, in situ hybridization and LSC. We have also examined the transportation of exogenous DNA into oocytes by immunofluorescene via PCR. Sperm cells were incubated with DNA/liposome complexes (1:4 ratio) in fertilization medium with BSA or without BSA. In situ hybridization demonstrated that the transfection rate of sperm cells with and without BSA was 41 and 68% respectively, when the cells were treated with liposome/DNA complexes and 13% for DNA alone. LSC analysis showed that the binding of exogenous DNA was greatly reduced by DNase I treatment which digests DNA bound onto spermatozoa, suggesting that some of the DNA was internalized into the sperm membrane. To find out whether transfected DNA was internalized into sperm intracytomembrane, sperm DNA was amplified by inverse PCR. No PCR products were detected from sperm cells, indicating that the foreign DNA was simply bound onto the sperm membrane. To investigate transfer rates of exogenous DNA into oocytes via sperm cells, we used immunofluorescene method to follow the distribution of foreign DNA via spermatozoa: a few exogenous DNA was located in the cytoplasm of early embryos (13/60, 21.7% for DNA+/liposome+/BSA) and was not located in the pronucleus and/or nucleus. These results suggest that most of the transfected sperm cells could carry the foreign DNA into the egg by in vitro fertilization, but that the transferred DNA is degraded in the developing embryos without stable integration into the zygote genome. Therefore, we have directly injected with transfected sperm cell into oocyte cytoplasm and observed that some of the exogenous DNA was detected in preimplantation embryonic cytoplasm and expressed at preimplantation stages, suggesting that exogenous DNA in early zygote has their integrity. In this study, we have not identified a noble mechanism that interfering transportation of foreign DNA into zygote genome via spermatozoa. Our data, however, demonstrated that inverse PCR and immunofluorescene methods would be used as a new tool for follow-up of gene distribution in oocyte via sperm cells.

• Proceedings of the KSAR Conference
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• 2002.06a
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• pp.91-91
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• 2002

There are many methods to introduce exogenous DNA into embryo for the purpose of producing transgenic animals. Exogenous gene can be integrated into oocyte as a form of sperm vector. In this study, sperm was used as a vector for transgene that is enhanced green fluorescent protein (EGFP). The objective of this study was to investigate the expression of exogenous gene in bovine embryos after injection of spermatozoa cocultured with EGFP fragment. Spermatozoa were plunged into liquid nitrogen and thawed several times or shaked in 0.2% Triton X-100 to remove sperm membrane which followed by DTT treatment. (omitted)

• Reproductive and Developmental Biology
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• v.30 no.3
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• pp.189-194
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• 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
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• v.13 no.11
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• pp.1514-1517
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• 2000

In chicken, exogenous genes introduced into germinal crescent region (GCR) of the early developmental stage, where primordial germ cells (PGCs) were concentrated, were successfully transferred to the gonads via PGCs. The foreign genes were also confirmed to be successfully incorporated into F1 and F2 generations. We tried to incorporate the exogenous genes into PGCs by lipofection, then the DNA mixture was injected into GCR at stage 3-5 or 9-11 of embryonic development (Hamburger and Hamilton, 1951). The manipulated eggs were incubated, and hatched chicks were reared until sexual maturation. F1 generation was obtained from the DNA-treated chicken (DNA-chicken) mated with normal birds. Furthermore, F2 generation was also obtained from the F1 chicken mated with normal birds. The transfer of introduced foreign genes were confirmed by marker gene detection methods and PCR analysis in the hatched chicks, F1 and F2 generations. However, in our experiments, DNA-chickens showed abnormal characteristics such as low egg production rate, abnormal appearance and decreased number of spermatozoa. In the case of F1 chicken, low egg production and the deterioration of sperm capacity for insemination in male chicken were observed.

• Proceedings of the Korean Society of Developmental Biology Conference
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• 2005.10a
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• pp.111-111
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• 2005

• Proceedings of the Korean Society of Embryo Transfer Conference
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• 2005.10a
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• pp.111-111
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• 2005

• Korean Journal of Animal Reproduction
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• v.26 no.3
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• pp.205-214
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• 2002

There are many methods to introduce exogenous DNA into embryo to produce transgenic animals. Exogenous gene can be integrated into oocyte by sperm vector. In this study, sperm was used as a vector for a transgene, which is encoding enhanced green fluorescent protein (EGFP). The objective of this study was to investigate the expression of exogenous gene in bovine embryos after injection of spermatozoa cocultured with EGFP DNA fragment. Spermatozoa were plunged into liquid nitrogen and thawed several times or shook in 0.2% Triton X-100 to remove sperm membrane followed by DTT treatment. The injected oocytes were co-cultured with vero cells in CR1aa, and expression of EGFP gene was observed under fluorescent microscope. Blastocyst formation rates of oocytes injected with sperm treated with DTT, DTT-freezing or DTT-Triton X-100 were 34.7, 39.4 and 31.9%, respectively. The rates of EGFP expression in oocytes injected with 54 ng DNA after DTT-treated, DTT-freezing and DTT-Triton X-100-treated sperm were 0, 19.1 and 13.9%. On the other hands, expression rate of oocytes injected with sperm cocultured with 13.5, 27 and 63.5 ng of EFGP DNA were 6.7, 9.0 and 5.1%, respectively. When intact sperm was mixed with 63.5 ng/${mu}ell$ EGFP DNA fragment, and then electroporated before injection, the expression rate of injected oocyte was 2%. Unexpectedly, electro-poration could not increase the expression rate. These results suggest that sperm can be used as a transgene vector, even if the efficiency was low (19.1%).

• Proceedings of the KSAR Conference
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• 2002.06a
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• pp.86-86
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• 2002

To examine the feasibility of using a sperm vector system for gene transfer, we have investigated the binding and the uptaking of foreign DNA into the sperm nucleus by PCR, in situ hybridization and LSC. We have also examined the transportation of exogenous DNA into oocytes by immunofluorescene via PCR. Sperm cells were incubated with DNA/liposome complexes (1:4 ratio) in fertilization medium with BSA or without BSA. (omitted)

• Asian-Australasian Journal of Animal Sciences
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• v.12 no.4
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• pp.520-524
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• 1999

The present experiments were designed to examine whether exogenous DNA injected into the germinal crescent region (GCR) of early stage of developing embryos, which is considered to be the main place from which PGCs originate, can be transferred to recipient chicken embryos. In this experiment, Miw Z (DNA) dissolved in the transfection reagent (TR: Boehringer, Germany) was introduced into the GCR of donor embryos at stage 3-5 or 9-11, followed by continued incubation until the stage 13-15 of embryonic development. The PGCs collected from the embryonic blood vessels were examined for the incorporation of the injected DNA into the PGCs by the methods of X-gal staining and PCR analysis. As the results, the foreign DNA was successfully incorporated into the PGCS, leading to their transfer to the gonadal tissues. The present results, therefore, suggest that the early stage (3-5 or 9-11) of chicken embryonic development would be more successful than stage 13-15 in transferring exogenous genes to the recipient embryos, leading to the possibility of producing transgenic chicken medianting the PGCS.