Lu J K, Chen T T, Allen S K, Matsubara T, Burns J C
Center of Marine Biotechnology, University of Maryland Biotechnology Institute, Baltimore, MD 21202, USA.
Proc Natl Acad Sci U S A. 1996 Apr 16;93(8):3482-6. doi: 10.1073/pnas.93.8.3482.
A pantropic pseudotyped retroviral vector containing the envelope protein of vesicular stomatitis virus was used as a gene transfer vector in the dwarf surfclam, Mulinia lateralis. These pantropic retroviral vectors have an extremely broad host cell range and can infect many nonmammalian species. Newly fertilized dwarf surfclam eggs were electroporated at 700 V in the presence of 1 x 10(4) colony-forming units of pantropic pseudotyped retroviral particles. Infection was well tolerated and did not affect the survival rate of the embryos. Gametes collected from P1 presumptive transgenic animals were analyzed for the presence of provirus by PCR, and in different experiments 13-33% of the gamete pools were positive for the transgene. Dot blot hybridization of DNA samples from the F1 offspring of two different crosses between infected P1 and wild-type individuals revealed that 28% and 31% of F1 offspring were transgenic, respectively. Southern blot analysis of DNA isolated from PCR-positive F1 animals confirmed integration of a single copy of the provirus into the host genome. Thus, the germ lines of these two P1 transgenic animals were mosaic for the transgene. Expression of beta-galactosidase encoded by the provirus was detected in transgenic but not control surfclam embryos. Pantropic pseudotyped retroviral vectors provide a useful method for the stable introduction of foreign genetic information into surfclams and may facilitate the introduction of desirable genetic traits into commercially important shellfish and crustaceans.
一种含有水泡性口炎病毒包膜蛋白的泛嗜性假型逆转录病毒载体被用作矮滨蛤（Mulinia lateralis）的基因转移载体。这些泛嗜性逆转录病毒载体具有极其广泛的宿主细胞范围，能够感染许多非哺乳动物物种。在1×10⁴个泛嗜性假型逆转录病毒颗粒集落形成单位存在的情况下，对新受精的矮滨蛤卵进行700V的电穿孔处理。感染耐受性良好，且不影响胚胎的存活率。通过PCR分析从P1推定转基因动物收集的配子中前病毒的存在情况，在不同实验中，13% - 33%的配子库对转基因呈阳性。对感染的P1个体与野生型个体之间两个不同杂交组合的F1后代的DNA样本进行斑点印迹杂交分析，结果显示分别有28%和31%的F1后代为转基因个体。对从PCR阳性的F1动物分离的DNA进行Southern印迹分析，证实前病毒的单拷贝整合到宿主基因组中。因此，这两只P1转基因动物的生殖系对于转基因是嵌合的。在转基因矮滨蛤胚胎而非对照胚胎中检测到由前病毒编码的β - 半乳糖苷酶的表达。泛嗜性假型逆转录病毒载体为将外源遗传信息稳定引入矮滨蛤提供了一种有用的方法，并且可能有助于将理想的遗传性状引入具有商业重要性的贝类和甲壳类动物中。
