• Clinical and Experimental Reproductive Medicine
• /
• 제14권1호
• /
• pp.51-59
• /
• 1987

The present experiments have been bone to verify the effects of the warming rate and the degenerated blastomere(s) on further development of the frozen and thawed 4- and 8-cell mouse embryos. The embryos obtained from the mouse superovulated and mated were frozen in the solution of 15M DMSO in PBS containing 10% FCS at a slowly cooling rate($0.3^{\circ}C/min$). Two methods of warming slowly($8^{\circ}C/min$) and quickly ($450^{\circ}C/min$) were applied for thawing embryos. The thawed embryos were grouped according to the number of healthy blastomere(s) in the embryos. Some of the embryos were eliminated their degenerated blastomere(s) by means of a micromanipulation technique. The embryos were examined their developmental phases after 48 or 72 hrs incubation. The rates of blastocyst development from the frozen and thawed 4- and 8-cell embryos were 72.7% and 73.5%, respectively in case of thawing slowly, and were 78.9% and 80.0%, respectively in case of thawing quickly. The rate in case of thawing quickly was significantly higher than that in case of thawing slowly. The rates of blastocyst development from the frozen and thawed 4- and 8-cell embryos eliminated their degenerated blastomere(s) increased 5.9% and 24.4%, respectively compared with those of control groups not eliminated. The more number of degenerated blastomere(s) were eliminated from the embryos, the higher rate of blastocyst development was shown. It may be concluded from the results that the quickly thawing method is better for increasing survival rate than the slowly thawing one, and that the degenerated blastomere(s) in the frozen and thawed embryos affects as an interfering factor for further development of the embryos.

• 한국동물번식학회:학술대회논문집
• /
• 한국동물번식학회 2004년도 춘계학술발표대회
• /
• pp.292-292
• /
• 2004

배아의 성별 판별을 위해 할구를 biopsy하여 핵상에서 정밀 분석을 하였다. 이 실험에서는 8-에서 16-세포기 배아의 할구를 배아 성결정의 대표물로 사용하여 IVF소 배아를 분석 평가하였다. 55개의 배아를 PCT후 biopsy하여 분석하였다. PCR에 의한 성판별에서 biopsy한 single blastomere와 blastocyst의 성판별의 일치하는 비율은 80%인 것으로 나타났다. IVF 수정란을 염색체 상태에서 평가하기 위해 8- 16- 세포기의 할구를 Karyotyping 하였다. 할구의 Karyotyping을 위해 metaphase 상태에서 vinblastine sulfate에 계속적으로 노출시켜 metaphse Ⅱ 상태를 유도하였다. (중략)

• 한국발생생물학회:학술대회논문집
• /
• 한국발생생물학회 2003년도 제3회 국제심포지움 및 학술대회
• /
• pp.92-92
• /
• 2003

Accurate analysis of nuclear status is needed when biopsied-blastomeres are used for embryo sexing. In this study, the nuclear status of blastomeres derived from 8- to 16-cell stage IVF bovine embryos was analyzed to evaluate the representative of single blastomere for embryo sexing. When 55 embryos were analyzed by PCR following biopsy, the coincident rate of sex determination between biopsied-single blastomere and matched blastocyst by PCR was 80 %. Karyotyping of biastomeres in 8- 16-cell stage bovine embryos was conducted to assess chromosome status of IVF embryos. To establish karyotyping of blastomeres, concentrations of vinblastine sulfate and duration of exposure time for metaphase plate induction with 8- to 16-cell stage bovine embryos were tested. The most effective condition for induction of metaphase plate (>45%) was 1.0 ug/ml vinblastine sulfate treatment for 15 h. In 22 embryos under the condition, only 8 embryos out of ten that had a normal diploid chromosome complement showed a sex-chromosomal composition of XX or XY (36.4%) and 2 diploid embryos showed mosaicism of the opposite sex of XX and XY in blastomeres of embryo (9.1%). One haploid embryo contained only one X-chromosome (4.5%). Four out of the other 11 embryos having a mixoploid chromosomal complement contained haploid blastomere with wrong sex chromosome (18.2%). These results suggested that morphologically normal bovine embryos derived from IVF had considerable proportion of mixoploid and sex-chromosomal mosaicism which could be the cause of discrepancies of the sex between biopsied-single blastomere and matched blastocyst by PCR analysis.

• 농업과학연구
• /
• 제38권2호
• /
• pp.269-275
• /
• 2011

The objective of this study was to develop a rapid and reliable PCR method for sexing of morula or blastocyst stage bovine embryo. BOV97M and bovine 1.715 satellite DNA sequences were selected for amplification of male and bovine specific DNA, respectively. But the unbalanced number of copies of these two repetitive sequences required some modification of PCR method. Karyotyping of blastomeres were carried for the confirmation of sex determination in bovine embryos. The coincidence rate of sex between biopsied-single blastomere and matched blastocyst was 80.0%. When in vivo- and in vitro- derived embryos were compared, 61.8% and 56.7% were male in in vitro- and in vivo-derived embryos, respectively. In vivo-derived embryos showed better hatching rate than in vitro-derived embryos following biopsy of blastomeres. In conclusion, rapid and effective PCR could be applied to sexing of bovine preimplantation embryos using single blastomere. The sensitivity of this assay may eliminate the need for biopsy of more than one nucleated blastomere and reduce trauma to the embryos derived from biopsy procedure.

• 한국수정란이식학회지
• /
• 제10권2호
• /
• pp.171-175
• /
• 1995

This study was performed to establish the condition and the methods for the techniques of insertion the isolated blastomere cells into cytoplasm, in order to research the develop-mental ability of bovine embryo blastomere cells in vitro produced. After 24h in vitro ovary maturation with the ovaries from a slaughter house, in vitro fertilization was performed to the vital sperms which their mobility were decided by percoll gradient method, with 2~8 cell stage embryos, the blastomeres were isolated in $Ca^2$+. $Mg^2$+-free PBS, and following that embedded into agar and alginate solution, respectively. The rates of in vitro develop-ment are as follows ; in agar embedded 11 among 120(9.2%) 1 /2~1 /3 blastomers cleaved and 6 among 93(6.5%) 1 /4~1 /8 blastomeres cleaved. In sodium alginate-embedded 14 among 84(16.7%) 1 /2~1 /3 blastomeres cleaved and 6 among 85(7.1%) 1 /4~1 /8 blastomeres cleaved. In case of Na-alginate, the rate of the cells were better than those of agar. The results suggest that the techniques for embeeding the isolated blastomeres into gel may help cloning of bovine early embryo without nuclear transplantation.

• 한국동물생명공학회지
• /
• 제35권4호
• /
• pp.323-328
• /
• 2020

Direct injection of genome editing tools such as CRISPR/Cas9 system into developing embryos has been widely used to generate genetically engineered pigs. The approach allows us to produce pigs carrying targeted modifications at high efficiency without having to apply somatic cell nuclear transfer. However, the targeted modifications during embryogenesis often result in mosaicism, which causes issues in phenotyping founder animals and establishing a group of pigs carrying intended modifications. This study was aimed to establish a genomic PCR and sequencing system of a single blastomere in the four-cell embryos to detect potential mosaicism. We performed genomic PCR in four individual blastomeres from four-cell embryos. We successfully amplified target genomic region from single blastomeres of 4-cell stage embryo by PCR. Sanger sequencing of the PCR amplicons obtained from the blastomeres suggested that PCR-based genotyping of single blastomere was a feasible method to determine mutation type generated by genome editing technology such as CRISPR/Cas9 in early stage embryos. In conclusion, we successfully genotyped single blastomeres in a single 4-cell stage embryo to detect potential mosaicism in porcine embryos. Our approach offers a simple platform that can be used to screen the prevalence of mosaicism from designed CRISPR/Cas9 systems.

• 한국수정란이식학회지
• /
• 제10권2호
• /
• pp.105-113
• /
• 1995

This study was conducted to examine the efficiency of enucleation and blastomere isolation from recipient oocytes and donor embryos, respectively and to determine the effect of oocyte age and electric voltage on the fusion rate and in vitro development of the fused oocytes in rabbit nuclear transplantation. Immature oocytes collected from ovarian follicles were matured in vivo for 12 h in TCM-199 containing FCS and hormones and in vivo matured oocytes were collected 17 to 18 h post-HCG. The fresh and frozen donor embryos of 8- to 16-cell stage were collected from the oviduct of superovulated does. The proportion of successfully enucleated oocytes was greatly lower in in vitro matured oocytes (42.3%) than that (62.7%) in in vivo matured oocytes The level of cytochalasin B for in vivo matured oocytes did not affect the efficiency of enuleation, but 7.5 $\mu$g /mL cytochalasin B for in vitro matured oocytes showed a high enucleation rate significantly. The isolation efficiency of a single blastomere nucleus did not differ between 8- and 16-cell stage embryos. The percentage of single blastomeres isolated from 16-cell stage fresh embryos after 0.5% pronase treatment was greatly higher at 16-min treatment (94.4%) than at 8-min(78. 1%) and the blastomeres(61.5%) isolated from frozen-thawed embryos after 16-min pronase were significantly fewer than those of fresh embryos. The age of recipient oocytes affected nuclear fusion rate. The reconstituted oocytes fused at 24-h age showed slightly higher fusion rate (77.8%) than those (65.0%)fused at 18-h age. The fusion rate of in vitro and in vivo matured oocytes inserted with fresh blastomere did not differ among electric voltages, but the cleavage rate and development to morula-blastocysts of in vitro matured oocytes was more higher under 0.6 kV/cm than under 0.8 to 1.2 kV/cm, while the cleavage rate and development of in vivo matured oocytes was higher under 0.8 to 1.0 kV/cm than under 1.2 kV/cm. The fusion and cleavage rate fol1owing insertion with frozen-thawed blastomere was not different between the in vitro and in vivo matured oocytes and was similar to those from fresh blastomere insertion.

• 한국동물번식학회:학술대회논문집
• /
• 한국동물번식학회 2004년도 춘계학술발표대회
• /
• pp.213-213
• /
• 2004

Exogeneous DNA can reproducibly be delivered by co-injected spermatozoa and this transgenesis method is very efficient protocol. But, mosaic patterns of transgenic embryos and offspring were shown frequently. Whole blastomere integration is important in transgenic animal economics. (omitted)

• 한국동물학회지
• /
• 제34권3호
• /
• pp.389-393
• /
• 1991

To obtain monozygotic multiplets from 8-cell mouse embryos, we artificially constructed chimeric embryos by introducing one blastomere (donor) of 8-cell embryos of Fl hybrid (C57BL/6 X CBA) mice into 4-cell ICR mouse embryos (carrier) of which one blastomere had been previously removed with a micromanipulator. After 42 h of culture, the developmental frequency of chimeric embryos to normal morula and blastocyst was 95% (310/328). When chimeric embryos at morula or blastocvst stage were transferred to pseudopregnant mice,39%, (70/180) of them were born. Most of the offspring (56/70) were the carrier type in coat color, whereas only three of them were the donor type, of which ho were assumed to be derived from single 8-cell donor embryo. Because the two donor type mice Ivere the same sex and produced only the donor type offspring from a testcross, they are probably monozvgotic multiplets of 8-cell mouse embryos. However, since their internal chimerism was not able to be examined, it remains to be determined if their genetic constitutions are identical.

• 한국동물학회지
• /
• 제33권1호
• /
• pp.45-52
• /
• 1990

성장중인 포유동물의 난자에 존재하는 성숙억제요인이 배아의 난하레 미치는 효과를 세포융합방법을 사용하여 조사하였다. 생쥐에서 성장중인 난자와 간기에 있는 2세포기 할구와 1 : 1로 융합하여 배양했을 때 60% 이상의 융합체들이 두개의 핵을 간직하고 있었으며 4세포기의 할구와 융합했을 때에는 90% 이상이 두개으 핵을 간직하고 있었다. 같은 조건으로 배양한 융합되지 않은 할구들이나 단독으로 배양한 할구들은 한 주기의 난할을 일으키었다. 이에대해 이미 유사분열기로 들어간 후기 2세포기 할구와 성장중인 난자와 융합을 했을 때에는 오히려 난자의 핵붕괴와 함께 염색체의 응축이 일어났다. 쥐의 성장중인 난자와 간기에 있는 생쥐 2세포기 할구와 1 : 1로 융합했을 때에도 역시 거의 모든 융합체들이 핵을 간직하고 있어서 분열이 정지되어 있었다. 이러한 결과는 생쥐나 쥐의 성장중인 난자에는 배아의 난할을 억제하는 성질이 있음을 보여주는 것이며 이미 분열기로 들어간 배아의 세포질에는 효과를 나타내지 못한다는 것을 보여주고 있다.