Guignot F, Perreau C, Cavarroc C, Touzé J-L, Pougnard J-L, Dupont F, Beckers J-F, Rémy B, Babilliot J-M, Bed'Hom B, Lamorinière J M, Mermillod P, Baril G
INRA, UMR85 Physiologie de la Reproduction et des Comportements CNRS, Nouzilly, France.
Reprod Domest Anim. 2011 Aug;46(4):656-63. doi: 10.1111/j.1439-0531.2010.01724.x. Epub 2010 Dec 1.
The objective of this study was to test the accuracy of genotype diagnosis after whole amplification of DNA extracted from biopsies obtained by trimming goat embryos and to evaluate the viability of biopsied embryos after vitrification/warming and transfer. Whole genome amplification (WGA) was performed using Multiple Displacement Amplification (MDA). Sex and prion protein (PRNP) genotypes were determined. Sex diagnosis was carried out by PCR amplification of ZFX/ZFY and Y chromosome-specific sequences. Prion protein genotype determination was performed on codons 142, 154, 211, 222 and 240. Embryos were collected at day 7 after oestrus and biopsied either immediately after collection (blastocysts and expanded blastocysts) or after 24 h of in vitro culture (compacted morulae). Biopsied embryos were frozen by vitrification. Vitrified whole embryos were kept as control. DNA of biopsies was extracted and amplified using MDA. Sex diagnosis was efficient for 97.4% of biopsies and PRNP genotyping was determined in 78.7% of biopsies. After embryo transfer, no significant difference was observed in kidding rate between biopsied and vitrified control embryos, whereas embryo survival rate was different between biopsied and whole vitrified embryos (p = 0.032). At birth, 100% of diagnosed sex and 98.2% of predetermined codons were correct. Offspring PRNP profiles were in agreement with parental genotype. Whole genome amplification with MDA kit coupled with sex diagnosis and PRNP genotype predetermination are very accurate techniques to genotype goat embryos before transfer. These novel results allow us to plan selection of scrapie-resistant genotypes and kid sex before transfer of cryopreserved embryo.
本研究的目的是检测从修剪山羊胚胎获得的活检组织中提取的DNA经全基因组扩增后基因型诊断的准确性，并评估玻璃化/复温及移植后活检胚胎的活力。使用多重置换扩增（MDA）进行全基因组扩增（WGA）。确定性别和朊病毒蛋白（PRNP）基因型。通过PCR扩增ZFX/ZFY和Y染色体特异性序列进行性别诊断。对密码子142、154、211、222和240进行朊病毒蛋白基因型测定。在发情后第7天收集胚胎，并在收集后立即（囊胚和扩张囊胚）或体外培养24小时后（致密桑葚胚）进行活检。活检后的胚胎通过玻璃化冷冻。将玻璃化的完整胚胎作为对照。提取活检组织的DNA并使用MDA进行扩增。97.4%的活检组织性别诊断有效，78.7%的活检组织确定了PRNP基因分型。胚胎移植后，活检胚胎与玻璃化对照胚胎的产羔率无显著差异，而活检胚胎与完整玻璃化胚胎的胚胎存活率不同（p = 0.032）。出生时，100%的诊断性别和98.2%的预先确定密码子是正确的。后代PRNP谱与亲本基因型一致。使用MDA试剂盒进行全基因组扩增并结合性别诊断和PRNP基因型预先确定是在移植前对山羊胚胎进行基因分型的非常准确的技术。这些新结果使我们能够在冷冻保存胚胎移植前计划选择抗瘙痒病基因型和羔羊性别。
