Svingen Terje, Wilhelm Dagmar, Combes Alexander N, Hosking Brett, Harley Vincent R, Sinclair Andrew H, Koopman Peter
Division of Molecular Genetics and Development, Institute for Molecular Bioscience, The University of Queensland, Brisbane, Australia.
Dev Dyn. 2009 Apr;238(4):956-64. doi: 10.1002/dvdy.21919.
Gene function during mouse development is often studied through the production and analysis of transgenic and knockout models. However, these techniques are time- and resource-consuming, and require specialized equipment and expertise. We have established a new protocol for functional studies that combines organ culture of explanted fetal tissues with microinjection and magnetically induced transfection ("magnetofection") of gene expression constructs. As proof-of-principle, we magnetofected cDNA constructs into genital ridge tissue as a means of gain-of-function analysis, and shRNA constructs for loss-of-function analysis. Ectopic expression of Sry induced female-to-male sex-reversal, whereas knockdown of Sox9 expression caused male-to-female sex-reversal, consistent with the known functions of these genes. Furthermore, ectopic expression of Tmem184a, a gene of unknown function, in female genital ridges, resulted in failure of gonocytes to enter meiosis. This technique will likely be applicable to the study of gene function in a broader range of developing organs and tissues.
在小鼠发育过程中，基因功能的研究通常通过构建和分析转基因及基因敲除模型来进行。然而，这些技术耗时且耗费资源，还需要专业设备和专业知识。我们建立了一种新的功能研究方案，该方案将离体胎儿组织的器官培养与基因表达构建体的显微注射和磁诱导转染（“磁转染”）相结合。作为原理验证，我们将cDNA构建体磁转染到生殖嵴组织中，作为功能获得性分析的手段，并将shRNA构建体用于功能缺失性分析。Sry的异位表达导致雌性向雄性的性逆转，而Sox9表达的敲低则导致雄性向雌性的性逆转，这与这些基因的已知功能一致。此外，在雌性生殖嵴中异位表达功能未知的基因Tmem184a，导致生殖母细胞无法进入减数分裂。这项技术可能适用于更广泛的发育器官和组织中的基因功能研究。
