• Proceedings of the Plant Resources Society of Korea Conference
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• 2003.10a
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• pp.59-67
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• 2003

An efficient plant regeneration system was developed for Hordeum vulgare L. cv Baegdong - an important Korean cultivar. The protocol was based on a series of experiments involving the sizes of mature embryos and the culture media. The embryo size is found to be critical for the establishment of embryogenic callus. Embryos of 1.1-1.5 mm size showed a much higher ability to produce embryogenic callus capable of regenerating green plants. The auxins picloram and dicamba proved effective in inducing callus from mature embryos. $2.5\;m;I^{-1}$ dicamba and $4.0\;mg\;I^{-1}$ picloram in Murashige and Skoog's (MS) medium was optimum for the induction of primary callus. The induced primary callus was loose and friable which ultimately developed into creamy white and compact callus after transferring into the fresh medium. Multiple shoots were induced in the MS medium supplemented with $6.0\;g\;I^{-1}$ maltose, $20\;mg\;I^{-1}$ sorbitol, $0.5\;mg\;I^{-1}$ 2, 4-D and $1.0\;mg\;I^{-1}$ kinetin and the rate was 6.5 shoots per embryo. Regenerated plants were hardy and developed roots rapidly in the medium containing $0.2\;I^{-1}$ IBA. This efficient plant regeneration system provides a foundation for generating transgenic plants of this important barley cultivar.

• Journal of Embryo Transfer
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• v.32 no.3
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• pp.227-233
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• 2017

Pig has been known to be one of the most feasible animals as a bioreactor to produce pharmaceuticals in milk and as a mediator in xenotransplantation research. Previously, we generated transgenic pigs for both purposes, which were expressing Factor 8, vWF, hTPA, and hEPO in milk, along with expression of MCP at GalT gene locus ($GalT^{-MCP/-MCP}$) as well as expressing MCP at GalT gene loci with CD73 expression ($GalT^{-MCP/+}/CD73$). In this study, we performed comparative analyses of sperm parameters between wild type male (WT) pig and those transgenic males to examine the effects of transgenes integrated into the pigs on motility, morphology, viability, and acrosome integrity of the spermatozoa. Our results showed that the rates of actively motile spermatozoa of WT, Factor 8, vWF, hTPA, hEPO, $GalT^{-MCP/+}/CD73$, and $GalT^{-MCP/-MCP}$ pigs were 85.0%, 83.3%, 82.5%, 83.3%, 82.5%, 77.5%, and 78.7%, respectively. Whereas, the rates of morphologically normal spermatozoa of WT, Factor 8, vWF, hTPA, hEPO, $GalT^{-MCP/+}/CD73$, and $GalT^{-MCP/-MCP}$ pigs were 90.0%, 80.0%, 80.0%, 83.3%, 85.0%, 91.8%, and 80.8%, respectively. In addition, the viability in spermatozoa of WT, Factor 8, vWF, hTPA, hEPO, $GalT^{-MCP/+}/CD73$, and $GalT^{-MCP/-MCP}$ pigs were 93.9%, 82.4%, 89.9%, 83.9%, 87.4%, 92.8%, and 83.6%, respectively. The rates of spermatozoa with normal acrosome integrity in WT, Factor 8, vWF, hTPA, hEPO, $GalT^{-MCP/+}/CD73$, and $GalT^{-MCP/-MCP}$ pigs were 98.1%, 98.6%, 98.6%, 98.7%, 98.1%, 99.5%, and 95.1%, respectively. There were no significant differences in motility, morphology, viability, and acrosome integrity of the spermatozoa among WT, Factor 8, vWF, hTPA, and hEPO, $GalT^{-MCP/+}/CD73$, and $GalT^{-MCP/-MCP}$ pigs. These mean that neither random integration nor targeted integration of the transgene into chromosome of pig effect on characteristics of spermatozoa. Ultimately, the transgenic male pigs subjected in this study could apply to propagate their progenies for production of human therapeutic proteins and advancing the xenotransplantation research.

• 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)

• Proceedings of the KSAR Conference
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• 2002.06a
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• pp.48-48
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• 2002

Chimerism has become an important tool for investigating fundamental aspects of early embryonic development and differentiation in mammals for producing transgenic animals. The objective of this study was to evaluate the developmental capacity of chimeric embryos reconstructed with parthenotes and IVF bovine embryos into empty zona pellucida. The MII oocytes were activated by two treatment groups [Group 1, 5 μM inomycin, 5min, ＋ 10 ㎍/㎖ cycloheximide (CHX)/5 ㎍/㎖ cytochalasin B (CCB), 3 h; Group 2, 5 μM ionomycin, 5 min ＋ 1.9 mM 6-dimetylaminopurine (6-DMAP), 3 h]. (omitted)

• Journal of Aquaculture
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• v.12 no.4
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• pp.275-281
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• 1999

The utility of RSV-LTR and carp beta-actin promoters was evaluated in a marine flatfish species, Limanda Yokohamae by examining successful expression of transgenic DNA in muscles (transfected by direct injection) and in early embryos (transformed by lipofected sperm). The expressed pattern of injected DNA in skeletal muscles was dependent on the DNA amount injected. The activity reached to maximal level at 48 hours post injection, and persisted up ot 1 month transiently. Gene transfer into early embryo of this species was successfully achieved using lipofected sperm with the efficiency ranging 36.8% to 48.1%. The expression of transgene during embryonic development was shown as stage-specific and transient.

• Proceedings of the PSK Conference
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• 2003.10b
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• pp.145.3-146
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• 2003

Transgenic Eleutherococcus senticosus plants were prepared by introducing the genes for squalene synthase (SQS), hygromycin phosphotransferase (HPT) and green fluorescent Protein (GFP) through Agrobacterium-mediated transformation. The enzyme, SQS, represents a putative branch point in the isoprenoid pathway capable of diverting carbon flow specifically to the biosynthesis of phytosterol and oleanolic acid. The full SQS gene was isolated from P. ginseng roots. Early globular embryo clusters developed from embryogenic callus were used as the explant source. (omitted)

• Proceedings of the KSAR Conference
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• 2001.10a
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• pp.48-49
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• 2001

While transgenic manipulation in mice have been very successful the same is not true for cattle and pigs. The inability to isolate ES cells from the bovine and porcine has precluded the utilization of the gene targeting technology in these species. Fortunately new advances in cloning by nuclear transfer have opened up a unique opportunity to undertake precise genetic modification in cattle and pigs. The ability of a number of different laboratory groups to successfully clone cattle is due to numerous research programs focused on nuclear transfer in cattle, and the enormous base of knowledge developed over the last 20 years involving the application of assisted reproductive techniques in cattle. Successful and repeatable procedures for in vitro oocyte maturation, in vitro fertilization, and in vitro embryo culture are now well established for cattle. In our laboratory we have utilized nuclear transfer to reproduce the genotypes of several animals, selected for cloning based on their inherent genetic value. Results that we have obtained to date are similar to those reported by other laboratories. (omitted)

• Korean Journal of Poultry Science
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• v.35 no.3
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• pp.241-245
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• 2008

Lentiviral vector system is efficient vehicles for the delivery of exogenous genes, and it is generally used in the generation of transgenic chickens. In this study, we used recombinant lentiviral vectors to generate transgenic chicks that express the human superoxide dismutase-3 gene driven by the chicken ovalbumin promoter. It is well known that superoxide dismutases(SODs) are believed to play a crucial role in protecting cells against oxygen toxicity. There are three forms of SOD proteins: cytosolic Cu-Zn SOD, mitochondrial Mn SOD, and extracellular SOD(SOD-3). The recombinant lentivirus containing the human SOD-3 gene was injected into the subgerminal cavity of freshly laid eggs. Subsequently, the embryos were incubated to hatch using phases II and III of the surrogate shell ex vivo culture system. From 341 injected embryos, the 78 chicks hatched after 21 days incubation. The hatched chicks were screened for the human SOD-3 gene by using PCR. Two of 47 male chickens that survived to sexual maturity contained the human SOD-3 gene in their semen. These results showed that our transgenic chicken generation system was completely established.

• Journal of Sericultural and Entomological Science
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• v.53 no.1
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• pp.55-60
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• 2015

Melittin is the main component of Bee Venom and has antibacterial activity against several bacteria. To produce the melittin antimicrobial peptide, we constructed transgenic silkworm that expressed melittin gene under the control BmA3 promoter using piggyBac vector. The use of the 3xP3-driven EGFP cDNA as a marker allowed us to rapidly distinguish transgenic silkworm. Mixtures of the donor vector and helper vector were micro-injected into 300 eggs of bivoltin silkworms, Baegokjam. In total, 131 larvae (G0) were hatched and allowed to develop into moths. The resulting G1 generation consisted of 36 broods, and we selected 4 broods containing at least 1 EGFP-positive embryo. The rate of successful transgenesis for the G1 broods was 11%. We identified 12 EGFP-positive G1 moths and these were backcrossed with wild-type moths. With the aim of identifying a melittin as antimicrobial peptide, we investigated the Radical diffusion Assay (RDA) and then demonstrated that melittin possesses high antibacterial activities against gramnegative bacteria.

• Korean Journal of Animal Reproduction
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• v.27 no.1
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• pp.69-75
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• 2003

The objective of this study is to improve the efficiency of embryo transfer in generation of transgenic mice using inbred mouse (C57BL/6J) embryos. The embryos of C57BL/6J and BCF1 mice were superovulated by the standard protocol. One-cell stage of embryos were microinjected and the resulted one-or two-cell were transferred into one-or two-side oviducts of foster mother, respectively. When one-cell stage of embryos were transferred into one-side oviduct of 0.75 d.p.c. foster mother, the results were not ideal because of showing pregnancy ratios of 68.8$\pm$7.83% for C57BL/6J and 48.3$\pm$14.22% for BCF1, and development ratios of pups vs transferred embryos of 11.9$\pm$5.51% for C57BL/6J and 10.5$\pm$8.03% fur BCF1 . However, when two-cell stage of embryos were transferred into two-side oviducts of 0.5 d.p.c. foster mother, we got better results of 94.4$\pm$9.64% and 100$\pm$0% pregnancy ratio, and 22.1$\pm$0.4% and 21.8$\pm$0.38% development ratio for C57BL/6J and BCF1, respectively. Therefore, transferring two-cell stage of C57BL/6J embryos into two-side oviducts of 0.5 d.p.c. foster mother may be competitive to the result in BCF1 embryos. Even if there are a lot of other factors affecting these results, we conclude from these experiments that transfer of two-cell embryos into two-side oviducts of 0.5 d.p.c. foster mother is a more efficient and safe method than others in generating transgenic mice using inbred mice embryos.