Mariol Marie-Christine, Walter Ludivine, Bellemin Stéphanie, Gieseler Kathrin
Université Claude Bernard Lyon.
J Vis Exp. 2013 Dec 9(82):e50773. doi: 10.3791/50773.
Microinjecting DNA into the cytoplasm of the syncytial gonad of Caenorhabditis elegans is the main technique used to establish transgenic lines that exhibit partial and variable transmission rates of extrachromosomal arrays to the next generation. In addition, transgenic animals are mosaic and express the transgene in a variable number of cells. Extrachromosomal arrays can be integrated into the C. elegans genome using UV irradiation to establish nonmosaic transgenic strains with 100% transmission rate of the transgene. To that extent, F1 progenies of UV irradiated transgenic animals are screened for animals carrying a heterozygous integration of the transgene, which leads to a 75% Mendelian transmission rate to the F2 progeny. One of the challenges of this method is to distinguish between the percentage of transgene transmission in a population before (X% transgenic animals) and after integration (≥75% transgenic F2 animals). Thus, this method requires choosing a nonintegrated transgenic line with a percentage of transgenic animals that is significantly lower than the Mendelian segregation of 75%. Consequently, nonintegrated transgenic lines with an extrachromosomal array transmission rate to the next generation ≤60% are usually preferred for integration, and transgene integration in highly transmitting strains is difficult. Here we show that the efficiency of extrachromosomal arrays integration into the genome is increased when using highly transmitting transgenic lines (≥80%). The described protocol allows for easy selection of several independent lines with homozygous transgene integration into the genome after UV irradiation of transgenic worms exhibiting a high rate of extrachromosomal array transmission. Furthermore, this method is quite fast and low material consuming. The possibility of rapidly generating different lines that express a particular integrated transgene is of great interest for studies focusing on gene expression pattern and regulation, protein localization, and overexpression, as well as for the development of subcellular markers.
将DNA显微注射到秀丽隐杆线虫合胞体性腺的细胞质中，是用于建立转基因品系的主要技术，这些转基因品系的染色体外阵列向下一代的传递率部分可变。此外，转基因动物是嵌合体，且在数量可变的细胞中表达转基因。可以使用紫外线照射将染色体外阵列整合到秀丽隐杆线虫基因组中，以建立转基因以100%传递率的非嵌合转基因品系。在这个程度上，对紫外线照射的转基因动物的F1后代进行筛选，以寻找携带转基因杂合整合的动物，这导致向F2后代的孟德尔传递率为75%。这种方法的挑战之一是区分整合前群体中转基因传递的百分比（X%转基因动物）和整合后（≥75%转基因F2动物）。因此，这种方法需要选择一个转基因动物百分比显著低于75%孟德尔分离率的非整合转基因品系。因此，通常首选染色体外阵列向下一代的传递率≤60%的非整合转基因品系进行整合，而在高传递品系中进行转基因整合则很困难。在这里，我们表明，当使用高传递转基因品系（≥80%）时，染色体外阵列整合到基因组中的效率会提高。所描述的方案允许在对表现出高染色体外阵列传递率的转基因蠕虫进行紫外线照射后，轻松选择几个具有纯合转基因整合到基因组中的独立品系。此外，这种方法相当快速且耗材少。快速产生表达特定整合转基因的不同品系的可能性，对于专注于基因表达模式和调控、蛋白质定位和过表达的研究，以及对于亚细胞标记的开发非常有意义。
