• 한국수정란이식학회지
• /
• 제27권1호
• /
• pp.63-69
• /
• 2012

The objective of this study was to investigate the effectiveness of cryopreservation methods for the effect of various vitrification containers, such as EM-grid, OPS, or cryo-loop on the survival and developmental rate of vitrified mouse pronuclear embryos, and mouse cleavage embryo, at 21, 24, 27 and 30 hr after hCG injection. Post-thaw cleavage was similar among treatments, while the developmental rates of mouse blastocyst and hatched blastocyst were higher ($p$ <0.05) in 27 hr and 30 hr than 21 hr. The developmental rate of hatched blastocyst at vitrified cleavage mouse embryos in cryo-loop was significantly higher than vitrified pronuclear embryos of control group as well as EM-grid and OPS ($p$ <0.05). The developmental rate using cryo-loop was higher than EM-grid, but in case of OPS at vitrified cleavage and mouse pronuclear embryos, no significant difference was noticed. These results of our study show that the developmental rates of mouse embryos were unaffected by various vitrification containers, but in case of mouse embryos and hatched blastocysts at late vitrified pronuclear embryos the developmental rates were higher than early vitrified pronuclear embryos. Moreover, the developmental rate of hatched blastocyst at vitrified cleavage mouse embryos was significantly higher than vitrified pronuclear embryos. For better execution of this study, it will be mandatory to include improvement of vitrification containers, cryopreservation methods and conditions, higher survival rate, safe preservation, contamination and embryo loss.

• Asian-Australasian Journal of Animal Sciences
• /
• 제22권2호
• /
• pp.174-180
• /
• 2009

This study was carried out to establish an appropriate condition for the efficient cryopreservation of the mouse pronuclear embryo. In vitro cryopreservation of pronuclear embryos was carried out by slow freezing or vitrification methods and development rate of 2-cell, blastocyst and hatched blastocyst was measured as well as survival rate of the thawed pronuclear embryo. After slow freezing, vitrification and thawing of mouse pronuclear embryos, the survival rate and blastocyst development rate for the vitrification group was 97.3 and 53.4%, respectively, which was significantly higher as compared to the slow freezing group with 88.6 and 23.9%, respectively (p<0.05). Blastocyst developmental rate in each experimental group was significantly higher for 21 h in the post-hCG group at 40.5-57.0% than the 24 h post-hCG group at 40.5% (p<0.05). ICM (Inner cell mass) cell numbers of blastocyst-stage embryos during the different stages of mouse pronuclear embryos, slow freezing and vitrification period in the control and vitrification groups were 22.1${\pm}$2.7 and 17.0${\pm}$3.1-22.0${\pm}$3.2, respectively; hence, the slow freezing group (10.2${\pm}$2.0) had significantly higher cell numbers than those of the other two groups (p<0.05). Trophoblast (TE) cell number in the control group, 65.8${\pm}$12.6, was significantly higher than in the slow freezing group, 41.6${\pm}$11.1 (p<0.05). The total cell numbers in the control group and 21 h post hCG group were 87.9${\pm}$13.6 and 81.8${\pm}$14.1, respectively, and were significantly higher than for the slow freezing group (51.8${\pm}$12.6; p<0.05).

• 한국수정란이식학회지
• /
• 제26권3호
• /
• pp.201-207
• /
• 2011

The present study was performed to investigate the survival and subsequent embryonic developmental rate of immature and mature oocytes after vitrification and pronuclear stage embryos after slow-freezing and vitrification. We have also tried to examine the dependency of concentrations (7.5, 15%) and exposure time (5, 10, 20 min) of ED cryoprotectant on developmental rate of pronuclear stage embryos. The developmental rates of 2-ce1l and blastocyst embryos at mature oocytes were significantly (p<0.05) higher than immature oocytes. After slow freezing, vitrification and thawing of pronuclear stage embryo, the survival and developmental rates of blastocysts and hatched blastocysts were significantly (p<0.05) higher after vitrification than after slow-freezing. On contrary, the developmental rates of 2-cell embryos were significantly (p<0.05) higher after slow freezing than after vitrification. The cryopreservation methods of pronuclear stage embryos vitrified by exposed to 7.5% ED solution for 5 minutes was significantly (p<0.05) higher than other experimental group. The results of our study suggest 1hat the developmental rates of mature oocytes have been more successful than immature oocytes during vitrification. Vitrification was more efficient than slow freezing in case of pronuclear stage embryos. The effective cryopreservation method of pronuclear stage embryos was vitrified by exposed to 7.5% ED solution for 5 minutes.

• Clinical and Experimental Reproductive Medicine
• /
• 제34권2호
• /
• pp.117-124
• /
• 2007

목 적: 본 연구는 생쥐 전핵시기 배아를 완만동결법과 유리화동결법으로 동결-융해 후 배아의 생존율과 성장률을 비교하고자 시행하였다. 연구방법: 과배란을 유도한 생쥐로부터 전핵시기 배아를 획득하여 10% SSS가 첨가된 HTF 배양액으로 약 1시간 동안 배양한 후 두 개의 전핵이 관찰되는 정상적인 형태의 배아만 선별하여 동결하였다. 동결방법으로는 1.5 M PROH에 0.1 M sucrose가 함유된 완만동결법과 40% ethylene glycol, 18% Ficoll, 0.5 M sucrose가 혼합된 EFS40 용액과 EM grid를 이용하여 이용한 유리화동결법을 실시하였다. 동결-융해 후 전핵시기 배아의 회수율, 생존을 및 부화 포배기로의 성장률과 부화율을 비교하였다. 결 과: 각각의 방법으로 동결-융해 후 24시간 동안 배양하였을 때 2-세포기까지의 성장률은 완만동결군이 59.1%이었고 유리화동결군이 77.0%로 두 군간에 유의한 차이를 보였고 (p<0.003), 48시간 동안의 배양에서도 완만동결군이 53.3%이고 유리동결군이 72.6%로 유의하게 유리화동결군에서 높은 수세포기까지의 성장률을 보였으며 (p<0.003), 72시간 배양하였을 때의 상실배로의 성장률 역시 완만동결군이 46.7%이고 유리화동결군이 67.3%로 유리화동결군에서 유의하게 높은 성장률을 보였다 (p<0.001). 융해 후 144시간 동안 배양하였을 때의 부화포배기로의 성장률은 완만동결군이 26.3%이고 유리화동결군이 43.4%로 유리화동결군에서 유의하게 높은 성장률을 보였다 (p<0.005). 결 론: 생쥐 전핵시기 배아의 동결보존에서 유리화동결법은 완만동결법 보다 시간이 단축되고 비싼 장비가 필요없어 경제적이고 간단했을 뿐 아니라 동결-응해 후 전반적으로 높은 생존율과 성장률을 나타내었다.

• 한국동물번식학회:학술대회논문집
• /
• 한국동물번식학회 2002년도 춘계학술발표대회 발표논문초록집
• /
• pp.58-58
• /
• 2002

Currently, the technique of pronuclear microinjection is the most successful and most widely-used method for producing transgenic animals. Among this technique, surperovulation and embryo transfer are the crucial steps for obtaining a large number of fertilized eggs and birth as much as potential founders from the transferred embryos. (omitted)

• 한국발생생물학회지:발생과생식
• /
• 제2권2호
• /
• pp.197-203
• /
• 1998

포유류 배아발생 중 DNA 메틸화는 세포분화와 유전자발현에서 중요한 역할을 하는 것으로 알려져 있다. 그러나, 생쥐 착상전 초기배아 발생 중 메틸화효소에 의해 유지되는 DNA 메틸화의 중요성과 자세한 기작은 잘 이해되고 있지 않다. 이 연구에서 DNA 메틸화의 역할에 관하여 알아보기 위하여, 성숙난자와 착상전 초기배아에서 DNA 메틸전이효소의 발현양상을 조사하였다. 이를 위해, DNA 메틸전이효소를 암호화하고 있는 cDNA에서 primer를 고안하였다. Primer의 정확도와 PCR조건의 적합화를 통하여, DNA MTase 전사물이 성숙난자와 착상전 초기배아에서 검출되었다. DNA MTase의 mRNA량은 성숙난자에서 가장 높으며, 전핵시기까지 비슷한 정도로 유지되었다. 이후 8-세포기까지 지속적으로 감소하다 상실기 배아에서 다시 검출되어 포배기까지 증가하는 양상을 보였다. 그리고, RNA polymerase II 억제제를 전핵시기 배아에 처리하여, 난자와 전핵시기 배아에 다량 존재하는 전사물이 모계유래인 것을 확인하였다. 결국, 난자와 전핵시기 배아에 상대적으로 다량 존재하는 DNA 메틸전이효소의 전사물은 아마도 착상전 초기배아에서 DHA 메틸화의 유지에 필요하며, 착상전 초기배아 발생에 있어서 유전자발현과 세포분화에 영향을 줄 것임을 시사하고 있다.

• Clinical and Experimental Reproductive Medicine
• /
• 제50권4호
• /
• pp.244-252
• /
• 2023

Objective: We evaluated the efficacy of the newly developed optimized in vitro culture (OIVC) dish for cultivating preimplantation mouse embryos. This dish minimizes the need for mineral oil and incorporates microwells, providing a stable culture environment and enabling independent monitoring of individual embryos. Methods: Mouse pronuclear (PN) zygotes and two-cell-stage embryos were collected at 18 and 46 hours after human chorionic gonadotropin injection, respectively. These were cultured for 120 hours using potassium simplex optimized medium (KSOM) to reach the blastocyst stage. The embryos were randomly allocated into three groups, each cultured in one of three dishes: a 60-mm culture dish, a microdrop dish, and an OIVC dish that we developed. Results: The OIVC dish effectively maintained the osmolarity of the KSOM culture medium over a 5-day period using only 2 mL of mineral oil. This contrasts with the significant osmolarity increase observed in the 60-mm culture dish. Additionally, the OIVC dish exhibited higher blastulation rates from two-cell embryos (100%) relative to the other dish types. Moreover, blastocysts derived from both PN zygotes and two-cell embryos in the OIVC dish group demonstrated significantly elevated mean cell numbers. Conclusion: Use of the OIVC dish markedly increased the number of cells in blastocysts derived from the in vitro culture of preimplantation mouse embryos. The capacity of this dish to maintain medium osmolarity with minimal mineral oil usage represents a breakthrough that may advance embryo culture techniques for various mammals, including human in vitro fertilization and embryo transfer programs.

• 한국수정란이식학회지
• /
• 제13권2호
• /
• pp.89-96
• /
• 1998

Anti-phospholipid antihodies (aPL) have important roles in various pregnancy complications such as recurrent miscarrige, growth retardation, placental abruption and stillbirth. However, their biological actions on preimplantation development of oocytes are still unclear. In this study, we investigated whether either aPL containing sera or phospholipids could affect in vitro fertilization and development of mouse oocytes. Sera used in this study were collected from three patients and the presence of aPL in the sera was confirmed by enzymatic-linked immunosorbent assay. When mouse oocytes were cultured in a serum-free, Chatot, Ziomek and Bavister (CZB) medium (Experiment 1), addition of aPL-containing sera (10%) to CZB medium did not. significantly (P>0.05) influence sperm penetration of oocytes. However, development to the blastocyst stage was significantly (P<0.05) inhibited by serum addition, and formation of morulae (16-23% vs. 58%) and blastocysts (0-4% vs. 21%) was markedly reduced compared with no addition (Experiment 2). In Experiment 3, pronuclear stage embryos were cultured for 96 h in GZB medium supplemented with 1 $\mu$g /ml phosphatidyl ethanolamine, 1 $\mu$g/ml phosphatidyl inositol or 1 $\mu$g /ml phosphatidyl choline. No increase in embryo development was found after addition of the phospholipids to CZB medium. These results suggest that 1) aPL have an inhibitory role in preimplantation development of mouse embryos, and that 2) the action of aPL may be related to a specific phospholid (s) rather than the tested phospholipids in the present study.

• 한국수정란이식학회지
• /
• 제30권3호
• /
• pp.207-211
• /
• 2015

미세주입법에 의한 형질전환 마우스 제작에는 대량의 전핵기란을 필요로 한다. 본 실험에서는 심플한 형질전환 마우스 제작방법을 확립하기 위해 초급속 동결한 전핵기란을 공시했다. 초급속 동결법으로 동결한 전핵기 체외수정란 139개를 융해 후 공시하고, ${\beta}-actin/luc^+$ 융합유전자를 미세주입하였다. 주입 조작 후, 형태학적으로 정상적인 수정란 101개(72.6%)를 5마리의 수란암컷 마우스에 이식하였다. 그 결과, 이식한 모든 마우스가 임신하고, 최종적으로 15마리(14.8%)의 산자가 태어났다. 한편, 450개의 체외수정란에 대해 동일한 배아조작 후에 338개(75.1%)가 생존하고 14마리의 수란암컷 마우스에 이식 하였다. 그 중에 78%의 수정암컷 마우스가 임신하고, 54마리(19.1%)의 산자가 태어났다. 태어난 산자에 대해서는 southern blot 법에 의해 염색체 내의 도입유전자의 도입을 확인한 결과, 동결수정란 처리구와 체외수정란구에서 각각 6.6%(1/15), 5.5% (3/54)의 마우스에서 도입유전자의 도입이 확인되었다. 더욱이 두 처리구 전부의 형질전환 마우스의 미부조직에서 도입유전자인 루시페라제 유전자의 발현이 관찰되었다. 이상의 결과에 의해 체외수정란 초급속 동결보존법을 사용한 Tg 마우스 제작방법의 확립을 확인하였고, 이러한 결과들로부터 실험기간의 단축과 작업의 간소화에 크게 이바지할 것으로 사료된다.

• 한국수정란이식학회지
• /
• 제30권4호
• /
• pp.299-303
• /
• 2015

KO mice provide an excellent tool to determine roles of specific genes in biomedical filed. Traditionally, knockout mice were generated by homologous recombination in embryonic stem cells. Recently, engineered nucleases, such as zinc finger nuclease, transcription activator-like effector nuclease and clustered regularly interspaced short palindromic repeats (CRISPR), were used to produce knockout mice. This new technology is useful because of high efficiency and ability to generate biallelic mutation in founder mice. Until now, most of knockout mice produced using engineered nucleases were C57BL/6 strain. In the present study we used CRISPR-Cas9 system to generate knockout mice in FVB strain. We designed and synthesized single guide RNA (sgRNA) of CRISPR system for targeting gene, Abtb2. Mouse zygote were obtained from superovulated FVB female mice at 8-10 weeks of age. The sgRNA was injected into pronuclear of the mouse zygote with recombinant Cas9 protein. The microinjected zygotes were cultured for an additional day and only cleaved embryos were selected. The selected embryos were surgically transferred to oviduct of surrogate mother and offsprings were obtained. Genomic DNA were isolated from the offsprings and the target sequence was amplified using PCR. In T7E1 assay, 46.7% among the offsprings were founded as mutants. The PCR products were purified and sequences were analyzed. Most of the mutations were founded as deletion of few sequences at the target site, however, not identical among the each offspring. In conclusion, we found that CRISPR system is very efficient to generate knockout mice in FVB strain.