• 한국가축번식학회지
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• 제26권4호
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• pp.385-394
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• 2002

This study was constructed the correlations of the embryonic developmental rates and the frequency of chromosome aberration using ear-skin-fibroblast cell in nuclear transfer (NT) derived embryos. Karyoplast-oocyte complexes were fused and activated simultaneously, then cultured for seven days to assess development. The developmental rates of NT and in vitro fertilization (IVF) embryos were 55.4% vs 63.5%, 31.7% vs 33% and 13.4% vs 16.8% in 2 cell, 8 cell and blastocyst, respectively. Firstly, the frequency of chromosome aberrations were evaluated using fluorescent in situ hybridization (FISH) technique with porcine chromosome 1 submetacentric specific probe. Chromosome aberration was detected at day 3 on the embryo culture, the percentages of chromosomal aneuploidy in NT and IVF embryos at 4-cell stage were 40%, 31.3%, respectively. Secondly, embryonic fragmentation was evaluated at 4-cell stage embryo. Frequency of embryonic fragmentations was in 51.3% of NT, 61.3% of IVF, 28.9% of parthenogenetic activation at 4-cell stage. The proportion of fragmentation in NT embryos was higher than activation embryos. This result indicates that chromosomal abnormalities and embryonic fragments are associated with low developmental rate in porcine NT embryo. It is also suggest that abnormal porcine embryos produced by NT related with lower implantation rate, increased abortion rate and production of abnormal fetuses.

• 한국수정란이식학회지
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• 제16권2호
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• pp.73-78
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• 2001

본 연구는 한우 체외수정란에 외래 유전자를 미세현미 주입한 후 체외 배발달을 조사하였다. DNA 미세주입은 체외수정 18~20시간 후에 DNA를 미세주입하였으며 체외 배발달율은 7일간 배양 후 조사하였다. 미세현미 주입 후 난할율은 36.3%로 대조구의 난할율 66.4% 보다 유의적으로 낮았으며(p<0.05) DNA가 주입된 수정란 중 상실배와 배반포배까지 발달율은 각각 5.6%와 1.9%로 대조구의 20.5%와 12.8%에 비하여 유의적으로 낮게 나타났다. 체외발달 배양액 내 L-ascorbic acid와 $\alpha$-tocopherol 첨가 배양시 상실배와 배반포배 발달율이 대조구에 비하여 유의적으로 높게 나타났다.(p<0.05) 따라서 미세현미 주입된 수정란의 체외 배발달 배양액에 항산화제의 첨가는 높은 체외 배발달율을 얻을 수 있으며 또한 체외성숙과, 체외 수정된 한우수정란을 이용하여 형질전환 한우 생산이 가능하리라 사료된다.

• 한국가금학회지
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• 제23권1호
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• pp.9-17
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• 1996

이 연구의 목적은 주입구의 위치에 따른 병아리 배아의 생존율을 서로 비교하고 가장 적합한 주입구 위치를 찾기 위하여 실시되었다. 멸균처리된 핀셋을 사용하여 난각의 첨단부와 둔단부 그리고 옆부분에 주입구를 각각 만들었다. 연구 결과, 둔단부에 주입구 (BE1)를 만든 수정란의 발생율이 가장 높았으나 내부난각막이 불투명하여 혈관내 미세주입이 어렵다. 따라서 본 연구에서는 첨단부에 주입구를 만든 다음 이 주입구를 통하여 약 2 $\mu$L의 DMEM 용액을 2.5일령된 배자의 혈관에 주입하였고, 주입부위의 출혈을 막기 위해 DMEM 용액을 주입한 후 공기방울을 넣은 결과 생존율이 약 17.0% 이었다. 따라서 이러한 주입구에 의한 방법은 생식세포가 조작된 germline chimera 또는 형질전환닭을 생산하는데 매우 유용한 시스템으로 이용될 수 있을 것이다.

• 한국동물번식학회:학술대회논문집
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• 한국동물번식학회 2001년도 춘계학술발표대회
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• pp.56-56
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• 2001

The exogenous gene transfer by intracytoplasmic sperm injection (ICSI) procedure has been recently used to produce transgenic mice and pigs. Sperm-mediated DNA transfer has the potential to markedly simplify the generation of transgenic animals. This method may serve as an alternative to the pronucleus injection of DNA for the production of transgenic pigs. Therefore, in this study, we investigated the expression of transgene after co-injection of spermatozoon or sperm head with green fluorescent protein (GFP) gene into in vitro matured porcine oocytes. Spermatozoon and sperm head, that was obtained by sonication, were treated with 0.03% Triton X-100 to remove the membrane. They were preincubated with linearized pEGFP-N1 for 1 min, and then embryos cultured NCSU23 medium for 2.5 days after co-injected of sperm and DNA. We monitored expression of GFP in embryos under epifluorescent microscope. The remove of sperm membrane did not alter the developmental competence of embryos after ICSI. At 7 days following injection, the rates of blastocysts following injection of intact sperm (15.0%), and of sperm with disrupted membrane (14.2%) were higher than that following IVF (10.0%). Porcine oocytes injected with sperm which co-cultured with DNA concentration of 1, 0.1, and 0.01 ng were 60, 65.7 and 75% and 18.5, 37.4 and 22.2% for rates of cleavage and GFP expression, respectively. In vitro matured porcine oocytes injected with sperm and isolated sperm head resulted in 69 and 59.7% of cleavage rates, respectively The rates of embryo GFP expressed did not significantly different between sperm (20.4%) and sperm head (20.0%) injection. The transgenic embryos with the clusters of positive blastomeres were observed under fluorescent microscope. Most of embryos expressed GFP gene showed mosaicism. They showed GFP expression at 1/4, 2/4 and 3/4 of blastomeres at the 4-cell stage. Among these 4-cell embryos, the expression rate of 1/4 blastomere group (54.6%) was higher than the other groups (15.3-30.7%). These results indicate that membrane disrupted sperm could attach with exogenous DNA, and that this procedure may be useful to introduce foreign gene into porcine oocytes. Therefore, our data suggest that the ICSI car be a useful tool to efficiently produce transgenic pig as well as other mammals.

• 한국동물번식학회:학술대회논문집
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• 한국동물번식학회 2004년도 춘계학술발표대회
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• pp.191-191
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• 2004

Transgenic animals are produced primarily by microinjecting exogenous DNA into the male pronuclei of a zygote. Microinjection method for gene transmitting is successful in mice but not efficient in farm animals, limiting it's general utility such as a large scale facility and labour. Based on our finding that sperm cells bind with exogenous DNA, sperm was used as a vector for producing transgenic animals to introduced green fluorescence protein(GFP) gene. (omitted)

• Asian-Australasian Journal of Animal Sciences
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• 제16권5호
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• pp.650-654
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• 2003

The accuracy of Polymerase chain reaction (PCR) assay in sexing of sheep embryos was assessed in this study. A total of 174 ovine embryos produced in vitro at different stages of development (2, 4-8 cell stages, morula and blastocyst) were sexed. The universal primers (P1-5EZ and P2-3EZ) used in this assay amplified ZFY/ZFX-specific sequences and yielded a 445 bp fragment in both sexes. Restriction enzyme analysis of ZFY/ZFX-amplified fragments with Sac I exhibited polymorphism between sexes, three and two fragments in males and in females, respectively. For verification of accuracy, blood samples of known sex were utilized as positive controls in each test. The mean percentages of sex identification by this method at 2 cell, 4-8 cell, morula and blastocyst were $73.00{\pm}5.72$, $89.77{\pm}3.79$, $3.33{\pm}8.08$ and $79.6{\pm}9.09$, espectively with the over all male to female ratio of 1:0.87. It is concluded that the ZFY/ZFX based method is highly reliable for the sexing of sheep embryos.

• Journal of Plant Biotechnology
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• 제41권1호
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• pp.50-55
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• 2014

Plant-based vaccines possess some advantages over other types of vaccine biotechnology such as safety, low cost of mass vaccination programs, and wider use of vaccines for medicine. This study was undertaken to develop the transgenic maize as edible vaccine candidates for humans. The immature embryos of HiII genotype were inoculated with A. tumefaciens strain C58C1 containing the binary vectors (V662 or V663). The vectors carrying nptII gene as selection marker and scEDIII (V662) or wCTB-scEDIII (V663) target gene, which code EIII proteins inhibite viral adsorption by cells. In total, 721 maize immature embryos were transformed and twenty-two putative transgenic plants were regenerated after 12 weeks selection regime. Of them, two- and six-plants were proved to be integrated with scEDIII and wCTB-scEDIII genes, respectively, by Southern blot analysis. However, only one plant (V662-29-3864) can express the gene of interest confirmed by Northern blot analysis. These results demonstrated that this plant could be used as a candidated source of the vaccine production.

• 한국발생생물학회지:발생과생식
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• 제11권3호
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• pp.219-226
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• 2007

본 연구는 생식선 키메라 생산효율을 높이기 위한 방법으로 busulfan 가온 주입법을 이용하여 효과적인 원시생식 세포의 이동능력을 검증하였다. 효율적인 생식선 키메라 닭 생산에서 중요한 요건 중 하나인 공여체 원시생식세포의 생존율을 측정한 실험에서는 시간이 지남에 따라 생존율에 변화를 보였으나, 평균 $70{\sim}80%$을 유지하고 있었으며, busulfan 처리 유무에 따른 공여체 원시생식세포 이동능력은 형광염색 후 주입한 실험에서 대조구가 4.8%인 반면 실험구는 23.5%을 나타냈다. 이식전 원시생식세포 배양 조건에 따라, 96시간과 118시간 배양 처리구에서 높은 이동능력을 보여 주었다. 원시생식세포의 형태학적, 생리학적 특징을 응용한 이식방법은 매우 효과적일 것이다. 그리고 본 연구에서는 생식반월의 발달단계 별 busulfan 처리 효과는 48시간이 가장 높은 53.4%였으며, 그러나 본 연구에서는 생식반월 유래 원시생식세포 이식은 48시간 이전, 혈관계가 발달하기 직전으로 가장 높은 효율을 보였다. 결론적으로 생식선 키메라 방법을 통한 형질전환 닭 생산 연구의 가장 큰 관건은 최대한 많은 수의 공여체 원시생식세포가 수용체의 저해작용 없이 안정적으로 수용체 gonad로 이동하여 분화하는 것으로, 본 연구 결과를 토대로 개선된 방법을 이용하면 높은 효율의 생식선 키메라 닭이 생산될 것으로 사료된다.

• Applied Biological Chemistry
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• 제38권5호
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• pp.387-392
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• 1995

대두 glycinin 유전자의 조직 특이적이고 분화 발달 특이적인 발현 조절 메카니즘을 연구하기 위하여 Gy2 유전자의 5' upstream 부위 염기서열을 조사한 결과, glycinin 유전자의 발현을 조절하는 인자로 여겨지는 여러 가지 조절 인자들을 발견하였다. 진핵세포 유전자에 공통적으로 존재하는 TATA box와 AGGA box가 존재하고, 종자 저장 단백질에서 공통적으로 발견되는 embryo factor binding sequence, RY repeat CACA sequence, ${\beta}$-conglycinin enhancer 와 유사한 sequence 등이 발견되었다. 이러한 조절 요소들이 Gy2 유전자의 발현 조절에 미치는 영향을 알아보기 위해 Gy2유전자의 5' upstream부위를 Exo III nuclease와 여러가지 제한효소를 이용하여 일련의 deletion mutants를 제조한 후 GUS 유전자와 결합시켰다. 이들 여러가지 chimeric constructs를 대두 원형질체에 전입하고 원형질체로부터 추출물을 분리하여 GUS 활성을 조사한 결과, $-28l{\sim}-223$ 혹은 $-l70{\sim}-122$ 부위를 포함하였을 경우 활성이 감소하였고, $-223{\sim}-170$ 혹은 $-l22{\sim}-16$ 부위를 포함하였을 경우 활성이 높게 나타났다. 이러한 Gy2 유전자의 이중적인 발현 양상은 glycinin 유전자의 발현조절에 음성 조절 요소와 양성 조절 요소가 관여하고있다는 사실을 제시해 주고 있다. 또한 이들 여러가지 chimeric constructs로 형질 전환된 담배의 종자와 잎에서 GUS활성을 조사한 결과, CaMV promoter를 포함하는 chimeric construct는 종자와 잎에서 모두 활성을 나타냈으나, Gy2 Promoter를 포함하는 chimeric constructs는 종자에서만 GUS 활성을 나타내고 잎에서는 활성이 나타나지 않는 조직 특이적인 발현 양상을 나타내었다.

• 한국수정란이식학회지
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• 제29권4호
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• pp.339-344
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• 2014

The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated protein (Cas9) system can be applied to produce transgenic pigs. Therefore, we applied CRISPR/Cas9 system to generate FoxN1-targeted pig parthenogenetic embryos. Using single guided RNA targeted to pig FoxN1 genes was injected into cytoplasm of in vitro matured oocyte before electrical activation. In results, regardless of the concentrations of vector, the cleavage rate were significantly (p<0.05) decreased ($4ng/{\mu}l$, 51.24%; $8ng/{\mu}l$, 40.88%; and $16ng/{\mu}l$; 45.22%) compared to no injection group (70.44%). The blastocyst formation rates were also decreased in vector injected 3 groups ($4ng/{\mu}l$, 7.96%; $8ng/{\mu}l$, 6.4%; and $16ng/{\mu}l$; 9.04%) compared to no injection group (29.07%). In addition, the blastocyst formation rates between sham injected group (13.51%) and no injection group (29.07%) also showed significant difference (p<0.05). The mutation rates were comparable between groups ($4ng/{\mu}l$, 18.4%; $8ng/{\mu}l$, 12.5%; and $16ng/{\mu}l$; 20.0%). The sequencing analysis showed that blastocysts derived from each group were successfully mutated in FoxN1 loci regardless of the vector concentrations. However, the deletion patterns were higher than the patterns of point mutation and insertion regardless of the vector concentrations. In conclusion, we described that cytoplasmic microinjection of FoxN1-targeted CRISPR/Cas9 vector could efficiently generate transgenic pig parthenogenetic embryos in one-step.