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

• Korean Journal of Agricultural Science
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• v.38 no.4
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• pp.681-687
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• 2011

Bioorgan transgenic cloned mini pig has a problem of growth retardation in uterus during end of pregnancy so that survival rate is very low in newborn piglet. In order to support their life after birth, cesarean section of fetus with sufficient growth in uterus was tested in this study. First of all, fetus growth measured using a ultrasound scanner during pregnancy in transgenic mini pig, comparing normal pig. After 113 days for delivering, fetus was removed out of uterus. Fetus growth for normal pig was 1.8 cm at 4weeks and 14.4 cm at end of pregnancy (15 weeks). At 113 days, fetus growth was $15.9{\pm}4$ cm in ultrasound scanner and real growth measurement from fetus removal out of uterus was $16.0{\pm}2$ cm. It is very a similar result between measurement of ultrasound scanner and real measurement. Therefore, using ultrasound scanner for measuring fetus growth will be useful to predict fetus growth in uterus.

• Asian-Australasian Journal of Animal Sciences
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• v.21 no.8
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• pp.1127-1133
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• 2008

This study was designed to develop a method of liquid storage of boar sperm at $4^{\circ}C$ by using the modified Beltsville F5 (BF5) diluent with bovine serum albumin (BSA) and N-acetyl-D-glucosamine. Boar sperm were stored in lactose-egg yolk and N-acetyl-D-glucosamine (LEN), BF5 and Golden-Pig liquid 4 (GPL4) diluents at $4^{\circ}C$ for 5 days and were examined for sperm viability, adenosine triphosphate (ATP) and in vitro fertilization (IVF). The percentage of sperm viability in GPL4 diluent was higher than in LEN and BF5 diluent from 1 to 5 days of storage at $4^{\circ}C$. The percentage of sperm viability steadily declined from 1 to 5 days of storage in the three different diluents. Sperm ATP in GPL4 diluent was higher than in LEN and BF5 diluents from 1 to 5 days of storage. Sperm ATP rapidly declined after 5 days of storage in the three different diluents. Porcine oocytes matured in vitro were inseminated with different sperm concentrations of liquid semen stored for 3 days in GPL4 diluent. The percentage of monospermic oocytes did not show any differences from 2.5 to $20{\times}10^5$ sperm/ml. However, the percentage of polyspermic oocytes in the sperm concentration of $2.5{\times}10^5$ sperm/ml was lower than in concentrations of 5, 10 and $20{\times}10^5$5 sperm/ml. The percentage of blastocysts from the cleaved oocytes at $2.5{\times}10^5/ml$ sperm concentration was significantly lower than at 5, 10 and $20{\times}10^5sperm/ml$ concentrations. In conclusion, GPL4 diluent can be stored at $4^{\circ}C$ for 5 days and showed higher sperm viability and sperm ATP concentration compared with LEN and BF5 diluents. Also, we found that GPL4 diluent can be used for IVF of porcine oocytes.

• Asian-Australasian Journal of Animal Sciences
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• v.19 no.12
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• pp.1761-1768
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• 2006

In this study, the comparative effects of transgenic corn (Mon 810 and Event 176) and isogenic corn (DK729) were investigated for their influence on in vitro rumen fermentation. This study consisted of three treatments with 0.25 g rice straw, 0.25 g of corn (Mon810/Event176/DK 729) mixed with 30 ml rumen fluid-basal medium in a serum bottle. They were prepared in oxygen free conditions and incubated at $39^{\circ}C$ in a shaking incubator. The influence of transgenic corn on the number of bacterial population, F. succinogenes (cellulolytic) and S. bovis (amylolytic), was quantified using RT-PCR. Fermentative parameters were measured at 0, 2, 4, 8, 12 and 24 h and substrate digestibility was measured at 12 and 24 h. No significant differences were observed in digestibility of dry matter, NDF, ADF at 12 and 24 h for both transgenic and isogenic form of corns (p>0.05) as well as in fermentative parameters. Fluid pH remained unaffected by hybrid trait and decreased with VFA accumulation as incubation time progressed. No influence of corn trait itself was seen on concentration of total VFA, acetic, propionic, butyric and valeric acids. There were no significant differences (p<0.05) in total gas production, composition of gas (methane and hydrogen) at all times of sampling, as well as in NH3-N production. Bacterial quantification using RT-PCR showed that the population number was not affected by transgenic corn. From this study it is concluded that transgenic corn (Mon810 and Event 176) had no adverse effects on rumen fermentation and digestibility compared to isogenic corn. However, regular monitoring of these transgenic feeds is needed by present day researchers to enable consumers with the option to select their preferred food source for animal or human consumption.

• Biomolecules & Therapeutics
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• v.14 no.2
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• pp.96-101
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• 2006

Amyloid$\beta(A{\beta})$ has been reported have an effect on the induction of oxidative stress that involves the functional and structural abnormalities in Alzheimer's disease. As a role of a radical scavenger, a green tea treatment was found have some inhibitory effect on the neurodegenerative process. The aim of this study was to determine if green tea catechin (GTC) reduces in transgenic model. To test this, transgenic mice carrying neuronspecific enolase (NSE) controlled C-terminus (105) of APP (APP-C105) were created and treated them with a low ana high dose of GTC for 6 months. Herein, we conclude that transgenic mice expressing NSE/APP-C105 were successfully created and the GTC-treated group exhibited significant reduction in body weight. Thus, GTC might be a good prevention of obesity or good treatment for AD patient.

• Proceedings of the Korean Society of Developmental Biology Conference
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• 1998.07a
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• pp.8-11
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• 1998

• Reproductive and Developmental Biology
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• v.32 no.2
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• pp.117-121
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• 2008

Our previous study showed that transgenic (TG) pigs harboring human EPO (hEPO) gene have been shown to have reproductive disorders, including low pregnancy rates, irregular estrus cycle and low little size. To investigate these reasons, we assessed estrus behavior (standing response) and plasma $17{\beta}$-estradiol ($E_2$) level, which partly reflect reproductive function, during the estrus cycles after synchronization and superovulation by hormone treatments. Then, we analysed blood composition and expression of hEPO gene in TG pigs. Pigs were injected with PG600. After 10 days, pigs were fed with Regumate porcine for 6 days. Blood samples were collected from jugular vein. Analysis of blood composition and $E_2$ level were measured by Hemavet 950 and $E_2$ ELISA kit, respectively. And, the expression of hEPO gene in reproductive organs was quantitated by real-time RT-PCR. The percentage of estrus behavior in TG was significantly decreased. Hematocrit (HCT), hemoglobin (Hb) concentration and red blood cell (RBC) number were significantly higher in TG than wild type (WT). On the other hand, high expression of hEPO gene in TG was observed in the mammary gland as well as in the uterus. Moreover, plasma $E_2$ level was significantly higher in TG than WT. These results suggest that nonspecific expression of hEPO gene in the other organs of TG may affect blood composition and plasma $E_2$ level, thereby causing reproductive disorders.

• Proceedings of the KSAR Conference
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• 2000.10a
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• pp.1-2
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• 2000

High production of milk and its components are necessary to allow maximal growth of developing piglets. In this study, transgenic pigs were produced containing the $\alpha$-lactalbumin gene, whose product is a potential limiting component in the production of milk. Two lines of transgenic pigs were produced to analyze the effects that overproduction of the milk protein $\alpha$-lactalbumin may have on milk production and piglet growth. Transgenic pigs were produced through microinjection of the bovine $\alpha$-lactalbumin gene. The gene construct contained 2.0 kb of 5 flanking region, the 2.0 kb coding region and 329 bp of 3 flanking region. Sows hemizygous for the transgene produced as much as 0.9 g of bovine $\alpha$-lactalbumin per liter of pig milk. The production of the bovine protein caused approximately a 50 % increase in the total $\alpha$-lactalbumin concentration in pig milk throughout lactation. The concentration of bovine $\alpha$-lactalbumin was highest on day 0 and 5 of lactation and decreased as lactation progressed. The ratio of bovine to porcine $\alpha$-lactalbumin changed during the sow's lactation. This ratio was 4.3 to 1 on day 0 of lactation, but by day 20 of lactation the ratio was 0.43 to 1. This suggested that the bovine transgene and the endogenous porcine gene were under slightly different control mechanisms. The higher level of total $\alpha$-lactalbumin present on day 0 of lactation was correlated with higher lactose percentage on day 0 in transgenic sows (3.8 %) as compared to controls (2.6 %) (P < 0.01). Although there was also a trend for higher lactose percentage in transgenic sows on day 5 and 10 of lactation, no significant differences were observed. These data suggest that $\alpha$-lactalbumin is limiting early in lactation of swine. Furthermore, higher concentrations of $\alpha$-lactalbumin early in lactation may boost milk output.

• Reproductive and Developmental Biology
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• v.29 no.4
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• pp.235-239
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• 2005

This study was conducted to test whether the transgenic cattle pass the transgene to their progeny through germ cells, and whether the transgene is expressed in the mammary gland of ransgenic cows. Two male ransgenic calves were born from IVF-derived embryos injected with bovine $\beta-casein/human$ lactoferrin fusion gene and then grew up to be reproducible. Semen was collected from a transgenic bull after 18 mon of age and then frozen. Bovine oocytes matured in vitro were fertilized with spermatozoa of the transgenic bull and cultured in $50\;{\mu}L$ drops of CRlaa medium supplemented with 3 mg/mL BSA. After 48 h of culture, cleaved embryos were determined for the presence of transgenes by DNA polymerase chain reaction (PCR). Proportion of transgene positives among bovine embryos fertilized with sperm of the transgenic bull was $20.9\%$ (28/134). One of transgenic bulls did not produce transgenic sperm. Out of 34 calves produced from recipient heifers inseminated with semen of the other bull, 3 $(8.8\%)$ were transgenic animals (2 females and 1 male). Thus, one transgenic bull showed a low transmission frequency below Mendelian levels in both the IVF-derived embryos and his progeny. It was demonstrated by Southern blot that copy numbers of the transgene in the transgenic progeny enhanced about 1.8 times as compared to those of the founder bull The results demonstrate that the transgenic bull carrying human lactoferrin gene could pass his transgene to the progeny through germ cells, although he is a germ-line mosaic.

• Asian-Australasian Journal of Animal Sciences
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• v.17 no.12
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• pp.1641-1646
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• 2004

The objective of this study was to generate transgenic mice expressing human resistin gene by using the tetraploidembryonic stem (ES) cell complementation method. Human resistin gene was amplified from human fetal liver cDNA library by PCR, cloned into $pCR^{(R)}$ 2.1 $TOPO^{(R)}$ vector and constructed in pCMV-Tag4C vector. Mammalian expression plasmid containing human resistin was transfected into D3-GL ES cells by Lipofectamine 2,000, and then after 10-12 days of transfection, the human resistin-expressing cells were selected with G418. In order to produce tetraploid embryos, blastomeres of diploid embryos at the two-cell stage were fused with two times of electric pulse using 60 V 30 $\mu$sec (fusion rate: 2,114/2,256, 93.5%) and cultured up to the blastocyst stage (development rate: 1,862/2,114, 94.6%). The selected 15-20 ES cells were injected into tetraploid blastocysts, and then transferred into the uteri of E 2.5 d pseudopregnant recipient mice. To investigate the gestation progress, two E 19.5 mused fetuses were recovered by Cesarean section of which one fetus was confirmed to contain human resistin gene by genomic DNA-PCR. Therefore, our findings demonstrate that tetraploid-ES mouse technology can be considered as a useful tool to produce transgenic mice for the rapid analysis of gene function in vivo.