• 한국작물학회지
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• 제49권1호
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• pp.61-68
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• 2004

A reproducible transformation system via optimized regeneration media for Korean rice cultivars was established using Agrobacterium tumefeciens LBA4404 (pSBM-PPGN; gusA and bar). Although japonica rice genotypes were easier to produce transgenic plants compared to Tongil type cultivars, transformation efficiencies were not always correlated with regeneration efficiencies of non-transgenic callus on the control medium. Regeneration efficiencies of Donganbyeo, Ilmibyeo, and Manchubyeo were over 50% in non-transgenic control, however, transformation efficiencies were significantly low when only sucrose was added to the media as a carbon source. However, the medium, MSRK5SS-Pr (or MSRK5SM-Pr), that contains $5\textrm{mgL}^{-1}$ kinetin, $0.5\textrm{mgL}^{-1}$ NAA, 2 % sucrose (or maltose), 3% sorbitol, and $500\textrm{mgL}^{-1}$ proline, was the most efficient not only for regeneration of non-transgenic callus but also for regeneration of transgenic callus in the presence of L-phosphinotricin (PPT). Average transformation efficiencies of 16 Korean rice cultivars were significantly enhanced by using the optimized medium from 1.5% to 5.8% in independent callus lines and from 2.9% to 19.4% in tromsgenic plants obained. Approximately 98.9% (876 out of 885) transgenic plants obtained on optimized media showed basta resistance. Stable integration, inheritance and expression of gusA and bar genes were continued by GUS assay and PCR and Southern analysis of the bar gene. With Pst1 digestion of genomic DNA of transgenic plants, one to five copies of T-DNA segment were observed; however, 76% (19 out of 25 transgenic plants) has low copy number of T-DNA. The transformants obtained from one callus line showed the same copy numbers with the same fractionized band patterns.

• 한국가금학회지
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• 제42권3호
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• pp.239-245
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• 2015

Transgenic animals have been widely used for developmental biology studies, as disease models, and even in industry such as transgenic bioreactor animals. For transgenic birds, quail has the great advantages of small body size, short generation time, and frequent egg production. To date, retroviral or lentiviral transduction has been used to generate transgenic quail for various purposes. However, the efficiency of transgenic offspring production with these methods is relatively low and viral vector usage has safety issues. Unfortunately, non-viral transgenesis has not been established in quail due to a deficiency of stem cell and germ cell culture systems. In this study, we established a direct in ovo lipofection method that could be used to create transgenic quail without germline-competent cells or viruses. To optimize the injection stage during embryo development, the liposome complex (containing piggyBacCMV-GFP and transposase plasmids) was introduced into an embryonic blood vessel at 50 hr, 55 hr or 60 hr. GFP expression was detected in various tissues (heart, kidney, liver and stomach) on day 12 of incubation under a fluorescence microscope. Additionally, GFP-positive cells were detected in the recipient embryonic gonads. In conclusion, the direct in ovo lipofection method with the piggyBac transposon could be an efficient and useful tool for generating transgenic quail.

• BMB Reports
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• 제39권1호
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• pp.61-67
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• 2006

Molecular co-suppression phenomena are important to consider in transgene experiments. Embryogenic cells were obtained from immature cotyledons and engineered with two different gene constructs (pHV and pHVS) through particle bombardment. Both constructs contain a gene conferring resistance to hygromycin (hpt) as a selective marker and a modified glycinin (11S globulin) gene (V3-1) as a target. sGFP(S65T) as a reporter gene was, however, inserted into the flanking region of the V3-1 gene (pHVS). Fluorescence microscopic screening after the selection of hygromycin, identified clearly the expression of sGFP(S65T) in the transformed soybean embryos bombarded with the pHVS construct. Stable integration of the transgenes was confirmed by polymerase chain reaction (PCR) and Southern blot analysis. Seeds of transgenic plants obtained from the pHV construct frequently lacked an accumulation of endogenous glycinin, which is encoded by homologous genes to the target gene V3-1. Most of the transgenic plants expressing sGFP(S65T) showed highly accumulation of glycinin. The expression of sGFP(S65T) and V3-1 inherits into the next generations. sGFP(S65T) as a reporter gene may be useful to increase the transformation efficiency of transgenic soybean with avoiding gene co-suppression.

• 한국동물번식학회:학술대회논문집
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• 한국동물번식학회 2002년도 춘계학술발표대회 발표논문초록집
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• pp.89-89
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• 2002

In this study, we examined transmission efficiency and expression level of the transgenes in the transgenic mice. The transgenic lines secreting a considerable amount of human lactoferrin(LF) thrombopoietin(TPO), interleukin-10(IL-10) into their milk were subjected to access the inheritance and maintenance of transgenic phenotype. They were bred through three generations. The transmission frequency for each generations(F9, F10, F11) of 3 lines was 38.03±10.43%(13/35), 48.33±3.76%(19/39) and 31.83±8.88%(9/28) in the LF line, 51.33±18.98%(20/38), 63.70±35.71%(12/20) and 29.57± 15.05%(8/26) in the TPO line, 38.27±17.74%(15/37), 47.47±29.88%(14/28) and 50.87±5.85%(14/28) in the IL-10 line, respectively. (omitted)

• 한국가축번식학회지
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• 제27권4호
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• pp.325-331
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• 2003

This study was performed to test the cellulose digestibility using the transgenic pigs harboring cellulose degradation gene D (CelD). After delivered offsprings between normal pig and transgenic swine, DNA was isolated from piglets tail for PCR analysis. In first generation, five out of 65 piglets showed CelD positive. Unfortunately, four CelD-positive pigs were died during growing, but one survived pig was used as a transgenic founder to produce F$_1$ descendents. Among 3 F$_1$ transgenic pigs produced, one died and the remaining two pigs were used to test the fiber digest efficiency. An assorted feed was composite of 5％ fiber with other ingredients. The feed of 3 kg per day was provided to the pigs including transgenic founders and littermate controls. The manure quantity was measured daily for a month, and all manures were dried for three days to analysis nitrogen, phosphate and fiber concentrations. The fiber digestion efficiencies of the transgenic F$_1$ pigs showed approximately 10％ higher than those of control pigs. Fiber digestion was not greatly improved in transgenic pigs as it had been expected approximately 30％. Nitrogen concentration of transgenic pig's manure was slowly decreased compare to the control pigs. Because there were only two transgenic pigs tested, a large number of transgenic pigs may be necessary to obtain more reliable data. Breeding of animals to obtain sufficient transgenic pigs subjected for a further study is on progress. Taken together, this study demonstrated successful production of transgenic pigs with increase of cellulose digestibility in the porcine feed.

• 한국임상수의학회지
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• 제16권1호
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• pp.163-176
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• 1999

The nuclear transplantation technique is known as the most potential and efficient method for producing large numbers of genetically identical animals from a single embryo and somatic cells. After Dolly was introduced in 1997, many scientists were amazed. A possibility came to a reality that live offspring could be produced with differentiated somatic cells from an adult animal. On the other side, many in the press and the sensationalists focused on the socially, ethically and scientifically unacceptable sides of the technology. In this article, the history, current status and prospects of the technological development of nuclear transplantation in mammals and its application to the production of cloned animals are described. For the efficient and successful production of cloned embryos by nuclear transplantation, the right selection, preactivation and micromanipulation of oocytes as capacious recipient cytoplasm, the adequate and benefitial preparation of multiple totipotent embryonic and somatic cells as donor nuclei, fusion of them and in vitro production of cloned embryos are very critical. Recently the overall efficiency of production of cloned embryos and offspring in livestock has been much improved. Cloning will also be a more efficient, faster and useful way of creating transgenic fetuses for gene therapies, gene pharming, organs for xenotransplantation by preselection and mass production of transgenic embryos and consequently improving the production efficiency in transgenic animals. Further technical development of nuclear transplantation will enable large-scale production of cloned livestock and in near future the commercial cloning of animals will become a reality.

• 한국자원식물학회:학술대회논문집
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• 한국자원식물학회 2018년도 추계학술대회
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• pp.52-52
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• 2018

Development of herbicide resistant transgenic legume plants through Agrobacterium-mediated transformation has been worked in many previous studied. Plant regeneration after infection is the important step to obtain successful transgenic plants. Many attempts try to find the optimum media condition for plant regeneration after infection. However, the transformation efficiency of legume plants is still low. In this study, regeneration of some Korean legume species including two soybean cultivars (Dawon and Pungsan) and pea have been done with organogenesis which is used various kind of explants such as cotyledonary-nodes in soybean and bud-containing tissue in pea. We developed the optimum media condition for plant regeneration regulators under Agrobacterium-mediated transformation using different kind and various concentration of plant growth. As the results, B5 medium containing 2 mg/L of 6-benzylaminopurine was selected in this study for the optimum plant regeneration media. The segments were inoculated with Agrobacterium suspension harbored an IG2 vector containing bar gene which confers resistance to phosphinotricin (PPT) in 3, 5 and 7 days. The transformation efficiency was achieved in Dawon 3.03 % and pea 1.46 % with co-cultivation period of 7 days which is showed a high number of GUS positive expression period.

• 한국수정란이식학회지
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• 제17권2호
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• pp.101-108
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• 2002

Human Prourokinase (proUK) offers potential as a novel agent with improved fibrin specificity and, as such, may offer advantages as an attractive alternative to urokinase that is associated with clinical benefits in patients with acute peripheral arterial occlusion. For production of transgenic cow as human proUK bioreacotor, we conducted this study to establish efficient production system for bovine transgenic embryos by somatic cell nuclear transfer (NT) using human prourokinase gene transfected donor cell. An expression plasmid for human prourokinase was constructed by inserting a bovine beta-casein promoter, a green fluorescent protein (GFP) marker gene, and human prourokinase target gene into a pcDNA3 plasmid. Cumulus cells were used as donor cell and transfected with the expression plasmid using the Fugene 6 as a carrier. To increase the efficiency for the production of transgenic NT, development rates were compared between non-transfected and transfected cell in experiment 1, and in experiment 2, development rates were compared according to level of GFP expression in donor cells. In experiment 1, development rates of non-transgenic NT embryos were significantly higher than transgenic NT embryos (43.3 vs. 28.4％). In experiment 2, there were no significant differences in fusion rates (85.4 vs. 78.9％) and cleavage rates (78.7 vs. 84.4％) between low and high expressed cells. However, development rates to blastocyst were higher in low expressed cells (17.0 vs. 33.3％), and GFP expression rates in blastocyst were higher in high expressed cells (75.0 vs. 43.3％), significantly.

• 한국수정란이식학회지
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• 제28권3호
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• pp.185-191
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• 2013

The purpose of this study is to improve production efficiency of vitrified-thawed transgenic bovine embryos. Transgenic bovine embryos were produced by injection of FIV-GFP lentiviral vector into perivitelline space of in vitro matured MII stage oocytes, and then in vitro fertilization. EGFP-expressing transgenic bovine blastocysts were cultured in serum-containing and serum-free medium. These blsatocysts were vitrified by pull and cut (PNC) container made with 0.25 cm plastic straw. Results indicate that total developmental rates of normal IVF embryo cultured in serum-containing and-free medium into blastocyst were not significantly different (22.3 vs 21.5%) and those of GFP-expressing transgenic bovine embryo into blastocyst showed no significant difference between serum-containing (13.9%) and-free medium (13.1%). However, developmental rate of GFP transgenic embryo was significantly (P<0.05) lower than its of normal IVF embryos. In additional study, we vitrified GFP transgenic normal bovine blastocysts using PNC vitrification method. Survival rate of vitrified-thawed GFP transgenic blastocyst (23.1%) was significantly (P<0.05) lower than its of normal blastocysts (68.9%). Although, survival rate of vitrified-thawed GFP transgenic blastocyst was lower than its of normal blastocyst, our result may suggested that PNC vitrification method is feasible to cryopreserve transgenic embryos. Our next plan will be the production of GFP express transgenic bovine derived from vitrified-thawed embryos using PNC method.

• 아시안잔디학회지
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• 제18권4호
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• pp.211-219
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• 2004

Transgenic zoysiagrass (Zoysia japonica cv. Zenith) plants have been obtained by particle bombardment of embryogenic callus with the plasmid pSMABuba, which contains hygromycin resistance (hpt) and bialaphos resistance (bar) genes. Parameters on DNA delivery efficiency of the particle bombardment were partially optimized using transient expression assay of a chimeric $\beta-glucuronidase$(gusA) gene driven by the CaMV 35S promoter. Stably transfarmed zoysiagrass plants were recovered with a selection scheme using hygromycin. Transgenic zoysiagrass plants were confirmed by PCR analysis with specific primer for bar gene. Expression of the transgene in transformed zoysiagrass plants was demonstrated by Reverse transcriptase (RT)-PCR analysis. All the tested transgenic plants showed herbicide BastaR resistance at the field application rate of $0.1\%-0.3\%$.