Su Baofeng, Shang Mei, Li Chao, Perera Dayan A, Pinkert Carl A, Irwin Michael H, Peatman Eric, Grewe Peter, Patil Jawahar G, Dunham Rex A
School of Fisheries, Aquaculture, and Aquatic Sciences, Auburn University, Auburn, AL, 36849, USA,
Transgenic Res. 2015 Apr;24(2):333-52. doi: 10.1007/s11248-014-9846-4. Epub 2014 Nov 4.
Channel catfish (Ictalurus punctatus) embryos were electroporated with sterilization constructs targeting primordial germ cell proteins or with buffer. Some embryos then were treated with repressor compounds, cadmium chloride, copper sulfate, sodium chloride or doxycycline, to prevent expression of the transgene constructs. Promoters included channel catfish nanos and vasa, salmon transferrin (TF), modified yeast Saccharomyces cerevisiae copper transport protein (MCTR) and zebrafish racemase (RM). Knock-down systems were the Tet-off (nanos and vasa constructs), MCTR, RM and TF systems. Knock-down genes included shRNAi targeting 5' nanos (N1), 3' nanos (N2) or dead end (DND), or double-stranded nanos RNA (dsRNA) for overexpression of nanos mRNA. These constructs previously were demonstrated to knock down nanos, vasa and dead end, with the repressors having variable success. Exogenous DNA affected percentage hatch (% hatch), as all 14 constructs, except for the TF dsRNA, TF N1 (T), RM DND (C), vasa DND (C), vasa N1 (C) and vasa N2 (C), had lower % hatch than the control electroporated with buffer. The MCTR and RM DND (T) constructs resulted in delayed hatch, and the vasa and nanos constructs had minimal effects on time of hatch (P < 0.05). Cadmium chloride appeared to counteract the slow development caused by the TF constructs in two TF treatments (P < 0.05). The 4 ppt sodium chloride treatment for the RM system decreased % hatch (P < 0.05) and slowed development. In the case of nanos constructs, doxycycline greatly delayed hatch (P < 0.05). Adverse effects of the transgenes and repressors continued for several treatments for the first 6 days after hatch, but only in a few treatments during the next 10 days. Repressors and gene expression impacted the yield of putative transgenic channel catfish fry, and need to be considered and accounted for in the hatchery phase of producing transgenically sterilized catfish fry and their fertile counterparts. This fry output should be considered to ensure that sufficient numbers of transgenic fish are produced for future applications and for defining repressor systems that are the most successful.
用靶向原始生殖细胞蛋白的绝育构建体或缓冲液对斑点叉尾鮰（Ictalurus punctatus）胚胎进行电穿孔。然后，对一些胚胎用阻遏化合物、氯化镉、硫酸铜、氯化钠或强力霉素进行处理，以阻止转基因构建体的表达。启动子包括斑点叉尾鮰的nanos和vasa、鲑鱼转铁蛋白（TF）、修饰的酿酒酵母铜转运蛋白（MCTR）和斑马鱼消旋酶（RM）。敲低系统有Tet-off（nanos和vasa构建体）、MCTR、RM和TF系统。敲低基因包括靶向5'端nanos（N1）、3'端nanos（N2）或死亡末端（DND）的短发夹RNA干扰（shRNAi），或用于过表达nanos mRNA的双链nanos RNA（dsRNA）。这些构建体先前已被证明可敲低nanos、vasa和死亡末端，阻遏物的效果各不相同。外源DNA影响孵化率（%孵化），因为除了TF双链RNA、TF N1（T）、RM DND（C）、vasa DND（C）、vasa N1（C）和vasa N2（C）之外，所有14种构建体的孵化率均低于用缓冲液电穿孔的对照组。MCTR和RM DND（T）构建体导致孵化延迟，vasa和nanos构建体对孵化时间的影响最小（P < 0.05）。在两种TF处理中，氯化镉似乎抵消了TF构建体导致的发育迟缓（P < 0.05）。RM系统的4 ppt氯化钠处理降低了孵化率（P < 0.05）并减缓了发育。对于nanos构建体，强力霉素极大地延迟了孵化（P < 0.05）。转基因和阻遏物的不利影响在孵化后的前6天持续存在于几种处理中，但在接下来的10天中仅存在于少数处理中。阻遏物和基因表达影响了推定的转基因斑点叉尾鮰鱼苗的产量，在生产转基因绝育鱼苗及其可育对应鱼苗的孵化场阶段需要加以考虑和核算。应考虑这种鱼苗产量，以确保生产出足够数量的转基因鱼用于未来应用，并确定最成功的阻遏系统。
