Development of embryo transfer system of bovine embryos sexed with PCR

PCR鉴定牛胚胎胚胎移植系统的研制

• 批准号: 05556049
• 负责人: YAMADA Masayasu
• 金额: $ 8.45万
• 依托单位: KYOTO UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Developmental Scientific Research (B)
• 财政年份: 1993
• 资助国家: 日本
• 起止时间: 1993 至 1995
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-05556049/
• 关键词: Embryo transfer; Embryo sexing; Bovine SRY cDNA; PCR method; Cryopreservation; IVM; IVF bovine embryos; ウシSRY遺伝子; 胚の性判別; ウシSRYの発現; 性判別胚の凍結保存; 性判別胚の移植; 遺伝子増幅法; 性分別; 移植システム; ウシ精巣決定遺伝子(bSRY); ウシ胚; 経膣採卵; 無血清培養

Our objective was to test the practical use of direct transfer of frozen-thawed bovine embryos sexed with PCR method using bovine Y chromosome specific DNA probes. First, to develop bovine Y-specific DNA probes, cloning of bovine sex determining region of Y gene (bSRY), which is supposed to induce male differentiation of gonad during bovine embryogenesis was designed. As a result, using cloning techniques, such as RT-PCR with human SRY sequence as primers and total RNA extracted from bovine testes, and 5'RACE,two different sequences of bSRYcDNA were determined, but both had the evolutionary-conserved HMG box sequence. Therefore, the difference of the sequence may be attributed to the circular structure of bSRY mRNA,as has been described in mouse Sry. It was moreover found that the primers derived from bSRYcDNA were useful for sexing of bovine embryos with PCR method. Next, the transfer of frozen-thawed bovine embryos sexed with PCR method was directed. When the embryos biopsied were frozen using a freezing medium containing PVP as a cryoprotectant, the survival rate of embryos after thawing was very high and after transfer of those embryos, we could produce efficiently youngs with predicted sex.

本研究旨在探讨用牛Y染色体特异性DNA探针PCR法鉴定牛胚胎性别后直接移植的实用性。首先，为了开发牛Y特异性DNA探针，设计克隆了牛Y基因的性别决定区（bSRY），该基因被认为在牛胚胎发育过程中诱导性腺的雄性分化。结果，利用克隆技术，如以人SRY序列为引物，从牛睾丸中提取总RNA的RT-PCR和5 'RACE，确定了两个不同的bSRYcDNA序列，但都具有进化保守的HMG盒序列。因此，序列的差异可能归因于bSRY mRNA的环状结构，如小鼠Sry中所述。根据bSRYcDNA序列设计的引物可用于牛胚胎性别鉴定。然后，对PCR法性别鉴定的牛胚胎进行冻融移植。用PVP作为冷冻保护剂的冷冻液冷冻活检后的胚胎，解冻后的胚胎存活率很高，移植后的胚胎，我们可以有效地产生具有预测性别的胚胎。

项目成果

Taniguchi, S.: "Hatching of bovine embryos clerived from in vitro fertilization in a cell-free, chemically defined medium." Jpn. J. Embryo Transfer. 17. 170-176 (1995)

Taniguchi, S.：“在无细胞、化学成分确定的培养基中体外受精孵化牛胚胎。”

Kim, K. S.: "Functional role of cumulus cells during maturation in development of in vitro matured and fertilized bovine oocytes" Theriogenology. 45. 278- (1996)

Kim, K. S.：“卵丘细胞在体外成熟和受精牛卵母细胞发育成熟过程中的功能作用”动物发生学。

Tominaga, K.: "Sex of IVM, IVF and IVC blastocysts produced discrimirately every donor cows" J. Reprod. Dev.(in press).

Tominaga, K.：“IVM、IVF 和 IVC 囊胚的性别对每头供体奶牛的产生都有区别”J. Reprod。

Takagi, M.: "Survival rate of frozen-thawed bovine IVF embryos in relation to exposure time using various cryoprotectants." Cryobiology. 30. 306-312 (1993)

Takagi, M.：“冻融牛 IVF 胚胎的存活率与使用各种冷冻保护剂的暴露时间的关系。”

Goto, H.: "Sexing of bovine embryos by PCR amplification of biopsied trophoblast cell and its clinicalapplication." Jpn.J.Embryo Transfer. 17. 19-26 (1995)

Goto, H.：“通过活检滋养层细胞的 PCR 扩增对牛胚胎进行性别鉴定及其临床应用。”