通过双原核显微注射提高转基因小鼠研制效率的实验研究

Higher Efficiency of Transgenic Mouse Production Achievedby Double-pronuclear Microinjection

目的建立高效的转基因小鼠制备技术,为开展遗传工程动物模型研究奠定技术基础。方法通过向小鼠受精卵原核中注入不同浓度的DNA分子,筛选最适注射用DNA浓度;将K14/hCTLA4-Ig基因表达载体分子通过显微注射分别导入小鼠受精卵雌、雄原核,并设立单原核注射对照组;利用输卵管腹壶部穿刺移植法将注射后的小鼠受精卵移植于同期发情的受体母鼠;利用PCR对出生的转基因首建小鼠进行筛选。结果最适DNA分子浓度为10ng/μl;在单、双原核注射组胚胎2细胞卵裂率分别为52.3%(132/253)和45.0%(108/240),差异有显著性(P<0.05);注射胚胎移植后体内存活率分别为18.1%(24/132)和16.7%(18/108),差异无显著性;转基因首建小鼠阳性率分别为3/24和5/18,转基因阳性小鼠占总注射胚胎的比例为1.2%(3/253)和2.08%(5/240),差异有极显著性(P<0.01)。结论尽管双原核注射对胚胎的2细胞卵裂率有一定影响,但通过双原核注射可有效提高转基因小鼠的制备效率。

Objective To establish an efficient technical platform for genetically-engineering animal model researches. Methods Using Kunming mice as the laboratory animals, DNA molecules （K14/hCTLA4-Ig expression vector） at a concentration of 10 ng/μl was transferred into male and female pronuclei of mouse eggs by double-pronuclear microinjection, while singlepronuclear microinjection was performed as control. Cleaved injected eggs were transferred into oviducts of synchronized recipient mice. Offsprings were screened by PCR. Results In the double-pronuclear microinjection group： the cleavage rate of injected eggs was 45.0 % （ 108/240）, significantly lower than that of single-pronuclear injection group （52.3 % ） ; the survival rate in vivo of transferred eggs was 18.5% （20/108）, with no significant difference compared to that of control group （16.7%）; the transgenic rate of founder mice and total injected eggs was 27.8 % and 2.08 % , respectively, which was significantly higher than that of control （ 12.5 % and 1.2% ）. Conclusion By double-pronuclear injection, transgenic mouse production efficiency can be effectively improved.