Hyttinen J M, Peura T, Tolvanen M, Aalto J, Jänne J
Department of Biochemistry and Biotechnology, University of Kuopio, Finland.
Mol Reprod Dev. 1996 Feb;43(2):150-7. doi: 10.1002/(SICI)1098-2795(199602)43:2<150::AID-MRD3>3.0.CO;2-Q.
We have developed a simple digestion-polymerase chain reaction (PCR) assay for a simultaneous transgene detection and sexing of pronucleus-injected bovine preimplantation embryos. Bovine embryos were microinjected with dam-methylated gene construct and cultured in vitro for 6-7 days after the injections. The developed blastocysts and compact morulae were bisected and the embryonic biopsies representing mainly trophoblasts were subjected to the digestion-PCR, while the biopsied embryos remained in culture. Embryonic DNA was released with proteinase K and the samples were digested with a Dpnl-Bal31 mixture before the PCR amplification of the transgene, bovine alpha S1-casein, and bovine Y-chromosome fragments in the same reaction. The whole assay from biopsy to electrophoresis took less than 6 hr. The digestion removed up to 50 fg of dam-methylated transgene copies (unintegrated or contaminants) and also a few hundred copies of contaminating PCR products from the embryonic samples. The digestion-PCR assay eliminated all transgene contaminations from noninjected blastocysts, which were exposed to the microinjection DNA during the stay in injection chambers, and reduced the amount of transgene-positive embryos among pronucleus-injected blastocysts as compared with unmodified PCR. Analysis of 486 microinjected bovine embryo biopsies in 13 separate experiments revealed that we were able to sex 398 (82%) of the biopsies and 77 (19%) of the biopsies were scored as transgene positive and 57 (14%) as transgene questionable. Upon reanalysis of 41 of the biopsied embryos, 38 (93%) of the embryos were observed to be transgene negative and 2 questionable in both assays and uneven distribution of transgene copies was observed in one embryo. The results from sexing were in accordance with biopsies and remaining embryos in 38 (93%) of the embryos.
我们开发了一种简单的消化 - 聚合酶链反应（PCR）检测方法，用于同时检测原核注射牛植入前胚胎的转基因并进行性别鉴定。将经母本甲基化的基因构建体显微注射到牛胚胎中，并在注射后体外培养6 - 7天。将发育成的囊胚和致密桑葚胚一分为二，取主要代表滋养层的胚胎活检组织进行消化 - PCR检测，而活检后的胚胎继续培养。用蛋白酶K释放胚胎DNA，在同一反应中对转基因、牛αS1 - 酪蛋白和牛Y染色体片段进行PCR扩增之前，先用Dpnl - Bal31混合物消化样品。从活检到电泳的整个检测过程耗时不到6小时。消化过程去除了高达50 fg的经母本甲基化的转基因拷贝（未整合或污染物），还从胚胎样品中去除了数百个污染性PCR产物拷贝。消化 - PCR检测消除了未注射囊胚中的所有转基因污染，这些未注射囊胚在注射室停留期间接触到了显微注射的DNA，并且与未改良的PCR相比，减少了原核注射囊胚中转基因阳性胚胎的数量。在13个独立实验中对486个显微注射的牛胚胎活检组织进行分析发现，我们能够对398个（82%）活检组织进行性别鉴定，77个（19%）活检组织被判定为转基因阳性，57个（14%）为转基因可疑。对41个活检胚胎进行重新分析时，在两次检测中观察到38个（93%）胚胎为转基因阴性，2个可疑，并且在一个胚胎中观察到转基因拷贝分布不均。性别鉴定结果与38个（93%）胚胎的活检组织和剩余胚胎一致。
