Menck M, Mercier Y, Campion E, Lobo R B, Heyman Y, Renard J P, Thompson E M
Unité de Biologie du Développement, INRA, Jouy-en-Josas, France.
Transgenic Res. 1998 Sep;7(5):331-41. doi: 10.1023/a:1008841222138.
Transgenesis in domestic species, as a research tool and in biotechnological applications, has been limited by the expense of producing transgenic offspring by standard microinjection techniques. A major factor is the inefficiency of maintaining large numbers of recipient females, when a high percentage of these carry nontransgenic fetuses. There are two approaches to reduce this cost, the fusion of transfected fetal fibroblasts with enucleated oocytes, and the screening of microinjected embryos for transgene integration in blastocysts, prior to transfer. Here, we develop a luminescent screening system to select transgenic bovine embryos. A transgene with scaffold attachment regions flanking the murine HSP70.1 promoter linked to firefly luciferase cDNA, was microinjected into pronuclei of in vitro produced zygotes. At the blastocyst stage, the transgene was induced by heat shock (45 degrees C, 15 min) and 4-6 h later, luciferase expression was analyzed by photon counting imaging. Screened blastocysts were transferred to recipients and day 50 fetuses or calves were analyzed by PCR and Southern blot for transgene integration. When nonluminescent blastocysts were transferred, transgene integration was never observed. Of 13 fetuses derived from luminescent blastocysts, 3 contained integrated transgenes that were functional in all tissues examined. Image analysis of the signal emitted by positive blastocysts revealed that 9 nontransgenic fetuses were obtained from blastocysts that exhibited a localized luminescent signal. On the other hand, 3 of 4 fetuses derived from blastocysts that emitted light over more than 70% of their surface were transgenic. Thus, by selecting luminescent blastocysts on the basis of both signal intensity and distribution, the number of recipient females required to produce transgenic offspring can be greatly reduced. Using this technique it should also be possible to improve the efficiency of transgenesis by microinjection through studies in which vector design and integration conditions are examined at the blastocyst stage.
在家畜物种中，作为一种研究工具和生物技术应用手段，通过标准显微注射技术生产转基因后代的成本限制了转基因技术的发展。一个主要因素是维持大量受体雌性动物的效率低下，因为这些雌性动物中很大比例携带的是非转基因胎儿。有两种方法可以降低成本，一是将转染的胎儿成纤维细胞与去核卵母细胞融合，二是在移植前对显微注射的胚胎进行筛选，以检测其在囊胚中的转基因整合情况。在此，我们开发了一种发光筛选系统来选择转基因牛胚胎。将一个带有支架附着区域的转基因显微注射到体外产生的受精卵原核中，该支架附着区域位于与萤火虫荧光素酶cDNA相连的小鼠HSP70.1启动子两侧。在囊胚阶段，通过热休克（45摄氏度，15分钟）诱导转基因表达，4至6小时后，通过光子计数成像分析荧光素酶的表达。筛选后的囊胚被移植到受体动物体内，在第50天对胎儿或小牛进行PCR和Southern印迹分析，以检测转基因整合情况。当移植非发光囊胚时，从未观察到转基因整合。在13个源自发光囊胚的胎儿中，有3个含有整合的转基因，这些转基因在所有检测组织中都具有功能。对阳性囊胚发出的信号进行图像分析发现，有9个非转基因胎儿来自显示局部发光信号的囊胚。另一方面，在4个表面发光超过70%的囊胚所衍生的胎儿中，有3个是转基因的。因此，通过根据信号强度和分布选择发光囊胚，可以大大减少生产转基因后代所需的受体雌性动物数量。使用这种技术，还应该能够通过在囊胚阶段研究载体设计和整合条件的实验来提高显微注射转基因技术的效率。
