Stocker K M, Brown A M, Ciment G
Department of Cell Biology & Anatomy, Oregon Health Sciences University, Portland 97201-3098.
J Neurosci Res. 1993 Jan;34(1):135-45. doi: 10.1002/jnr.490340114.
We describe here a new method for transferring genes into cells of the neural tube and neural crest of early avian embryos in vivo. Using the marker gene lacZ as an example, we infected dissected neural tubes from Hamburger-Hamilton stage 12-13 quail embryos with a replication-defective retrovirus carrying lacZ during a 2 hr period of exposure to the virus in culture. Infected neural tubes were then grafted into uninfected host chicken embryos in ovo and, after continued development for several days, the chimeric embryos were processed for beta-galactosidase histochemistry to identify the progeny of infected cells. We show that virus-infected neural tubes grafted isotopically into the trunk region of host embryos gave rise to cells of both the spinal cord and neural crest. Infected neural crest cells localized within dorsal root ganglia, sympathetic ganglia, peripheral nerves, and within the skin, where they were likely to give rise to melanocytes. These data are consistent with those using other cell marking techniques applied to the neural crest, indicating that retrovirus infection in culture, grafting, and beta-galactosidase expression has a neutral effect on neural crest cell migration and localization. These results indicate the heterospecific grafting of early avian tissues infected with retroviruses carrying foreign genes may be an effective strategy for testing the biological role of various gene products during development.
我们在此描述一种将基因导入早期鸡胚神经管和神经嵴细胞的新方法。以标记基因lacZ为例，我们在培养过程中将携带lacZ的复制缺陷型逆转录病毒感染来自汉堡-汉密尔顿12 - 13期鹌鹑胚胎的解剖神经管，感染时间为2小时。然后将感染的神经管移植到未感染的宿主鸡胚的卵内，在继续发育数天后，对嵌合胚胎进行β-半乳糖苷酶组织化学处理，以鉴定感染细胞的后代。我们发现，同基因移植到宿主胚胎躯干区域的病毒感染神经管产生了脊髓和神经嵴的细胞。感染的神经嵴细胞定位于背根神经节、交感神经节、外周神经以及皮肤内，在皮肤中它们可能产生黑素细胞。这些数据与使用其他应用于神经嵴的细胞标记技术的数据一致，表明培养中的逆转录病毒感染、移植以及β-半乳糖苷酶表达对神经嵴细胞的迁移和定位具有中性影响。这些结果表明，用携带外源基因的逆转录病毒感染的早期鸡胚组织的异种移植可能是测试发育过程中各种基因产物生物学作用的有效策略。
