• Asian-Australasian Journal of Animal Sciences
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• v.13 no.10
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• pp.1367-1372
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• 2000

The present study was conducted to evaluate the efficiency of transgenic rabbit production by DNA microinjection using EGFP (Enhanced Green Fluorescent Protein) gene. In this experiment EGFP coding sequences fused to CMV promoter were microinjected into rabbit one-cell embryos, and then GFP expression and gene integration were evaluated in preimplantation embryos and fetuses recovered on day 15 of pregnancy to determine efficiency of transgenic rabbit production. Effect of DNA concentration was also tested on development in vitro following microinjection and transgene integration in fetuses. Development of embryos in vitro was decreased by DNA microinjection, but the rates of pregnancy and implantation were not significantly affected by microinjection. As development progressed in vitro percentage of GFP expression in rabbit embryos was decreased, resulting GFP expression detected in 37.5% of blastocysts. The efficiencies for production of transgenic fetuses were 4.0% and 7.6%, respectively, when $10ng/{\mu}l$ and $20ng/{\mu}l$ of DNA concentration were microinjected. Transgenic fetuses were confirmed by GFP expression and PCR analysis of fetus genomic DNA. These results indicated that DNA microinjection itself damaged embryo development and DNA concentration affected the efficiency of transgenic rabbit production.

• Asian-Australasian Journal of Animal Sciences
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• v.20 no.4
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• pp.482-486
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• 2007

Basic objective of this research was to compare the milk yield and composition of New Zealand White transgenic rabbit females expressing recombinant human factor VIII (hFVIII) in mammary gland during lactation with that of non-transgenic rabbit females of the same age during 30 days of lactation. Transgenic founders were generated by the microinjection of foreign DNA (mWAP-hFVIII gene construct) into the egg. F1, F2 and F3 generations of transgenic rabbits were obtained after mating of transgenic founder rabbits with non-transgenic rabbits. The amount of milk rejected was measured by weight-suckle-weight method at $10^{th}$, $20^{th}$and $30^{th}$ day of lactation. Quality of milk (content of fat, protein, lactose, dry ash, and some minerals) from transgenic and non-transgenic rabbit was also determined. Comparison of milk yield, determined by weight-suckle-weight method, showed significantly higher (p<0.05) milk production at day 20 of first lactation in non-transgenic females, but on the same day of second lactation higher milk yield was measured in transgenic ones. Significantly higher (p<0.05) content of milk fat and protein was determined in transgenic milk whilst higher content of lactose was found in non-transgenic milk. The content of minerals (calcium, phosphorus, magnesium and sodium) did not differ in both experimental and control groups. Our results showed that milk yield and composition of transgenic rabbit females (mammary specific transgenic over-expression of hFVIII) over several generations is only slightly and transiently different from milk yield of non-transgenic females, which had no significant consequence on the litter size and viability.

• Reproductive and Developmental Biology
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• v.32 no.4
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• pp.249-253
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• 2008

Interspecies somatic cell nuclear transfer (iSCNT) is a valuable tool for studying the interactions between an oocyte and somatic nucleus. The object of this study was to investigate the developmental competence of in vitro-matured porcine oocytes after transfer of the somatic cell nuclei of 2 different species (goat and rabbit). Porcine cumulus oocytes were obtained from the follicles of ovaries and matured in TCM-199. The reconstructed embryos were electrically fused with 2 DC pulses of 1.1kV/cm for $30{\mu}s$ 0.3M mannitol medium. The activated cloned embryos were cultured in porcine zygote medium-3 (PZM-3), mSOF or RDH medium for 7 days. The blastocyst formation rate of the embryos reconstructed from goat or rabbit fetal fibroblasts was significantly lower than that of the embryos reconstructed from porcine fetal fibroblast cells. However, a significantly higher number of embryos reconstructed from goat or rabbit fetal fibroblasts cultured in mSOF or RDH, respectively, developed to the morular stage than those cultured in PZM-3. These results suggest that goat and bovine fetal fibroblasts were less efficacious than porcine-porcine cloned embryos and that culture condition could be an important factor in iSCNT. The lower developmental potential of goat-porcine and porcine-bovine cloned embryos may be due to incompatibility between the porcine oocyte cytoplasm and goat and bovine somatic nuclei.

• Journal of Embryo Transfer
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• v.14 no.1
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• pp.1-8
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• 1999

The efficiency of transgenic livestock animal production may be improved by early selection of transgenci preimplantation embryos. To examine the possibility of GFP gene as a non-invasive marker for the early screening of transgenic embryo, the GFP gene was microinjected into rabbit zygotes and the later stages of preimplantation embryos were examined for the expression of GFP. The presence of injected DNA was detected by PCR analysis and the expression of GFP was detected by observing green fluorescence in embryos under a fluorescent microscope. Out of 108 GFP gene-injected rabbit zygotes, seventy three(67.6%) were fluorescence-positive. When 11 fluroresecence-positive blastocysts were analyzed for the presence of GFP gene by PCR, 6(54.5%) were positive, and all of the 8 flrouescence-negative blastocysts were also negative by PCR. The results indicate that the screening of transgene in rabbit embryos by PCR analysis and GFP detection could be a promising method for the preselection of transgenic embryos.

• Journal of Embryo Transfer
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• v.15 no.2
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• pp.167-173
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• 2000

The principal objective of this study was to clone transgenic embryos in order to improve the efficiency of transgenic animal production by the combination of microinjection and nuclear transplantation techniques. Mature female New Zealand White rabbits were superovulated by eCG and hCG treatments, fllowed by natural mating. Zygotes were collected from the oviducts at 18∼22 h after hCG injection by flushing with D-PBS containing 5% fetal calf serum(FCS). Two to three picoliters of green fluorescent protein(GFP) gene wa microinjected into male pronucleus. The foreign gene-injected zygotes were cultured in TCM-199 or RD medium containing 10% FCS with a monolayer of rabbit oviductal epithelial cells in a 5% CO2 incubator. The morulae expressing GFP gene were selected and their blastomeres were separated for the use of nuclear donor. Following nuclear transplantation of fluorescence-positive morula stage blastomeres, 13 (21.3%) out of 61 fused oocytes developed to blastocyst stage and all of the cloned blastocysts expressed GFP. The results indicate that the screening of transgene in rabbit embryos by GFP detection could be a promisible method for the preselection of transgenic embryos. Also the cloning of preselected transgenic embryos by nuclear transplantatin could be efficiently applied to the multiple production of transgenic animals.

• Journal of Embryo Transfer
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• v.13 no.2
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• pp.97-106
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• 1998

The pronuclear injection of metallothionein-human growth hormone (MT-hGH) gene into rabbit zygotes was performed to establish in vitro developmental system and to detect the presence of the injected gene by nested PCR. Mature female New Zealand White rabbits were superovulated by eGG and hCG treatments. The rabbits were mated and the zygotes were collected from the oviducts 18-22 h after hCG injection by flushing with D-PBS. Two to three picoliters of MT-hGH gene was microinjected into male pronuclei. The foreign gene-injected zygotes were cultured in TCM-199 or RD mediurn containing 10% FCS with a monolayer of rabbit oviductal epithelial cefls in a 5% $CO_2$ incubator. The presence of injected DNA in rabbit embryos or blastomeres at different developmental stages .vas detected by a nested PCR analysis. The results are summarized as follows ; 1.The developmental rate of the MT-hGH gene-injected zygotes to blastocyst was significantly higher in TCM-199 medium (68.1%) than in RD medium (42.9%). 2.The gene injection into pronuclei at 18 or 22 hours post hCG treatment during pronuclear stage did not much affect on the in vitro development of the rabbit embryos. 3.The rate of gene-positive embryos detected by the nested PCR analysis was significantly decreased when they developed to blastocysts. The results indicate that the screening of transgene in rabbit embryos by nested PCR analysis could be a prornisible method for the preselection of transgenic embryos. Furthermore, the preselection of transgenic embryos would greatly reduce hoth the cost and effort of production of transgenic animals.

• Korean Journal of Pharmacognosy
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• v.31 no.2
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• pp.149-156
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• 2000

Atherosclerosis is emerging as one of the major causes of death in Korea as well as Western societies. In the present study; hypocholesterolemic and antiatherogenic effects of the ethanol extract of Allium victorialis Makino was investigated using the conventional rabbit and the cholesteryl ester transfer protein (CETP)-transgenic mouse model. Hypercholesterolemia was induced by feeding high cholesterol diet to the animals for 30 days and they were then fed with high cholesterol diet containing 0.5% of the A. victorialis extract for additional 30 (or 40) days. In the experiment using rabbits, treatment with the A. victorialis extract significantly decreased plasma total cholesterol, low density lipoprotein (LDL)-cholesterol, triglyceride levels and lipid peroxidation compared to those in the control group. Total cholesterol contents in the liver and the heart were also significantly decreased. Lipid staining of the aorta isolated from the rabbits showed that treatment with the A. victorialis extract decreased formation of atheromatous plaques on the intima of the aorta. In the experiment employing CETP transgenic mouse model, treatment with the A. victorialis extract decreased the levels of plasma total cholesterol and the tissue triglyceride levels in the heart. These results demonstrated that the ethanol extract of A. victorialis lowered serum cholesterol levels, tissue lipid contents and accumulation of cholesterol in the artery.

• Korean Journal of Animal Reproduction
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• v.22 no.4
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• pp.419-424
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• 1998

The present study was carried out to examine the efficiency of cloning of transgenic embryos by nuclear transfer(NT) using gene-injected rabbit embryos. The rabbit embryos at pronuclear stage were microinjected with methallothionein-human growth hormone(MT-hGH) gene and cultured to 8- and 16-cell in TCM-199 containing 10% FCS with a monolayer of rabbit oviductal epithelial cells in a 5% $CO_2$incubator. The recipient oocytes were collected from the oviducts 14~16 h after hCG injection. The oocytes were enucleated and activated with 5$\mu$M ionomycin and 2mM 6-dimethylaminopurine. Blastomeres form gene-injected embryos were transferred into the enucleated oocytes by micromanipulation. The nuclear transplant oocytes were electrofused and co-cultured with rabbit oviductal cells. Following 120 h of culture, blastocysts were prepared for gene analysis by polymerase chain reaction(PCR). In previous experiment, the rate of gene-positive embryos detected by the nested PCR analysis was significantly decreased while developing to blastocyst(25%)(Kang et al., 1998). The fusion rate of gene-injected blastomeres was significantly(P＜0.05) lower than non-injected blastomeres(66% vs 80%). However, the NT embryos that were derived from gene-injected donor embryos did not differ from control embryos in development to the blastocyst stage(39% vs 31%). Of the 43 NT blastocysts developed from the gene-injected donor embryos, twelve(28%) were positive for the injected DNA. The results indicate that NT with gene-injected embryos can be successfully used for cloning and multiplication of transgenic embryos, furthermore applicable to improvement of transgenic animal production.

• Reproductive and Developmental Biology
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• v.28 no.3
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• pp.141-145
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• 2004

Mucin coat is deposited on the embryos during passage through the oviduct in rabbit. When in vitro cultured blastocysts were transferred to the recipients, the lack of mucin coat might account in part for failure of pregnancy after transfer. The present study were carried out to investigate whether deposition of mucin coat were induced when in vitro cultured blastocysts were transferred to recipients. At 19 ～20 hours post-coitus one-cell embryos were collected by flushing oviducts. These embryos cultured for 72 hours were reached to blastocyst stage. And these blastocysts were transferred to the oviduct of asynchronized (one day later than the donors) and synchronized recipient. To confirm deposition of the mucin coat, blastocysts transferred to the oviduct were recovered at 24 and 48 hours after the transfer. Fifty eight percent of blastocysts recovered from uterus of asynchronous recipient at 24 hours after transfer and 92.9% of blastocysts recovered from uterus of synchronous recipient were 0～10 ㎛ of mucin coat thickness. And 11.8% of blastocysts of asynchronized recipients and 7.1% of blastocysts from asynchronized recipients were in 11～20 ㎛ of mucin coat thickness. When blastocysts were recovered from uterus at 48 hours after transfer, 87.0% of blastocysts from asynchronized recipients and 5.9% of blastocyst from synchronized recipients were in 0～10 ㎛ of mucin coat thickness. And 76.5% of blastocysts of synchronized recipients and 4.4% of blastocysts from asynchronized recipients were in 11～20 ㎛ of mucin coat thickness. From these results it is speculated that the low implantation rate of in vitro cultured rabbit blastocysts transferred to oviduct of recipient was caused by high degeneration of the embryo after transfer and inappropriate deposition of mucin coat.

• Reproductive and Developmental Biology
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• v.30 no.4
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• pp.229-234
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• 2006

We have generated transgenic mice that expressed mouse extracellular superoxide dismutase (EC-SOD) in their skin. In particular, the expression plasmid DNA containing human keratin K14 promoter was used to direct the keratinocyte-specific transcription of the transgene. To compare intron-dependent and intron-independent gene expression, we constructed two vectors. The vector B, which contains the rabbit -globin intron 2, was not effective for mouse EC-SOD overexpression. The EC-SOD transcript was detected in the skin, as determined by Northern blot analysis. Furthermore, EC-SOD protein was detected in the skin tissue, as demonstrated by Western blot analysis. To evaluate the expression levels of EC-SOD in various tissues, we purified EC-SOD from the skin, lungs, brain, kidneys, livers, and spleen of transgenic mice and measured its activities. EC-SOD activities in the transgenic mice skin were approximately 7 fold higher than in wild-type mice. These results suggest that the mouse overexpressing vector not only induces keratinocyte-specific expression of EC-SOD, but also expresses successfully functional EC-SOD. Thus, these transgenic mice appeared to be useful for the expression of the EC-SOD gene and subsequent analysis of various skin changes, such as erythema, inflamation, photoaging, and skin tumors.