Sex determination of early emvryos of cattle and fowl without employing chromosome analysis

克隆了鸡W染色体特异性XhoI家族重复DNA的0.7kb和1.1kb两个重复单位。这些重复单元由约21 bp序列的串联重复组成，在4%聚丙烯酰胺凝胶电泳中迁移异常缓慢，表明这些单元形成了弯曲的DNA结构。在允许60%碱基对错配的非严格杂交条件下，以_<32>p标记的0.7 kb重复单元为探针，从火鸡和野鸡的雌性基因组dna中选择并克隆了Xhoi家族相关和W染色体特异性重复单元：PUMG0401携带来自火鸡的psi -0.4 kb片段，PUPV0501携带来自野鸡的taqi -0.5 kb片段。使用克隆的鸡的0.7 kb单位，对这些性染色体特异性重复DNA克隆用于早期胚胎性别鉴定的可行性进行了测试。结果表明，用_<32>p标记探针对6日龄鸡个体胚的部分粗DNA进行点印迹杂交，可获得清晰的雌性特异性杂交信号。用生物素-11- dutp对XhoI 0.7 kb单元进行缺口翻译，并将其作为探针进行点印迹杂交，并用亲和碱磷酸酶法检测杂交形成，结果表明，仅100ng DNA就足以产生明显的雌性特异性颜色发育。同样的方法也适用于72h鸡胚细胞核的单核苷酸杂交。

Two repeating units, 0.7kb and 1.1kb, of the XhoI family repetitive DNA, which is specific to the W chtomosome of chicken were cloned. These repeating units consist of tandem repeat of about 21-bp sequence and migrate unusually slow in 4% ployacrylamide gel electrophoresis, suggesting that these units form curved DNA configuration. Undef the non-stringent hybridization conditions allowing the presence of about 60% base pair mismatches in the resulting hybrids and using the_<32>p-labeled 0.7-kb repeating unit as a probe, Xhoi family-related and W chromosome-specific repeating units were selected and cloned from the female genomic DNAs of turky and pheasant: PUMG0401 carrying a PstI-0.4-kb fragment from the turkey and PUPV0501 carrying a TaqI-0.5-kb fragment from the pheasant.Feasibility of employing these sex chromosome-specific repetitive DNA clones in sexing of early embryos was tested using the cloned 0.7-kb unit of the chicken. It was demonstrated that a cleat female-specific hybredization signal could be obtained when a crude DNA preparation isolated from a part of the indivisual 6 day chicken embryo was subjected to dot blot hybrisization with the_<32>p-labeled probe. When the XhoI 0.7-kb unit was biotinylated by nick translation with biotin-11-dUTP and used as a probe in the dot blot thbridization and the hybrid formation was deterced by an avidin-alkalinephosphatase method, ad little as 100ng DNA was proven to be sufficient to give clear demale-specific color development. Similar method was also shown to be applicable to in sity hybridization with the nuclei from the 72h chiecken embryos.