Peippo Jaana, Viitala Sirja, Virta Jouni, Räty Mervi, Tammiranta Niina, Lamminen Terttu, Aro Johanna, Myllymäki Hannu, Vilkki Johanna
MTT Agrifood Research Finland, Biotechnology and Food Research, Jokioinen, Finland.
Mol Reprod Dev. 2007 Nov;74(11):1373-8. doi: 10.1002/mrd.20731.
We report a method for multiplex genotyping of bovine embryo microblade biopsies. We have tested the reliability of the method and the viability of the embryos in vitro and in vivo. Two polymorphic gene markers (GHR F279Y and PRLR S18N) associated with milk production traits and one marker for sex diagnosis (ZFX/ZFY) were genotyped simultaneously with a method that combines nested PCR and allelic discrimination. To test the accuracy of genotyping, in the first experiment the genotypes of 134 biopsies from in vitro produced embryos were compared to genotypes determined from the corresponding embryos after biopsy. The method proved to be highly accurate as only in three cases (two for PRLR S18N and one for GHR F279Y) out of 395 genotypes the genotype was in disagreement between the two samples. The viability of similarly biopsied embryos was tested in parallel: after 24-hr culture 94.6% of embryos recovered in vitro. In the second experiment, a total of 150 in vivo-produced embryos were biopsied on Day 7 and genotyped. After the genotyping results were obtained on Day 8, female embryos were selected for transfer. From a total of 57 selected embryos 43 were transferred individually and 14 as pairs. After single embryo transfers, 19 recipients became pregnant and after embryo transfers in pairs one became pregnant. The success of genotyping was tested with the genotypes of donors and bulls and also from the hair samples of born calves. All calves were females and of the same genotypes determined from the biopsy.
我们报告了一种用于牛胚胎微刀片活检多重基因分型的方法。我们已经测试了该方法的可靠性以及胚胎在体外和体内的活力。与产奶性状相关的两个多态性基因标记（GHR F279Y和PRLR S18N）以及一个性别诊断标记（ZFX/ZFY）通过结合巢式PCR和等位基因鉴别法同时进行基因分型。为了测试基因分型的准确性，在第一个实验中，将134个体外生产胚胎的活检样本的基因型与活检后从相应胚胎确定的基因型进行比较。该方法被证明具有高度准确性，因为在395个基因型中，只有3例（PRLR S18N两例，GHR F279Y一例）两个样本的基因型不一致。同时对经类似活检的胚胎的活力进行了测试：体外培养24小时后，94.6%的胚胎得以恢复。在第二个实验中，总共150个体内生产的胚胎在第7天进行活检并进行基因分型。在第8天获得基因分型结果后，选择雌性胚胎进行移植。在总共57个选定的胚胎中，43个单独移植，14个成对移植。单胚胎移植后，19头受体母牛怀孕，成对胚胎移植后，有1头怀孕。通过供体和公牛的基因型以及出生小牛的毛发样本测试了基因分型的成功率。所有小牛均为雌性，且与活检确定的基因型相同。
