Abstract：The production of transgenic animals is an important tool for experimental and applied biology. Over the years, many approaches for the production of transgenic animals have been tried, including pronuclear microinjection, sperm-mediated gene transfer, transfection of male germ cells, somatic cell nuclear transfer and the use of lentiviral vectors. At present, the most effective method for the production of transgenic mice is the pronuclear injection technology. Microinjection of DNA into the pronuclei of zygotes is the simplest and most widely used method for generating transgenic (Tg) mice (Mus musculus). However, it is always associated with random integration of multiple copies of the transgene, resulting in unstable, low, or no transgene expression due to positional effects and/or repeat-induced gene silencing. The main disadvantage of this technique is that the efficiency is not high. In this paper, the injection needle diameter, concentration of exogenous gene, single-/double-pronuclear microinjection, embryo transfer and other major steps were optimized. The results showed that different concentrations of exogenous gene had a great impact on the positive rate of transgenic mice. When the injection of exogenous gene concentration in 1.00, 2.50, 3.00 and 4.00 μg/mL, the positive rates of transgenic mice were 0.67%, 14.9%, 30.6% and 21.4%, respectively. Among them, the exogenous gene concentration at 3.00 μg/mL was significantly higher than that of other groups (P＜0.01). In the single-/double-pronuclear microinjection test, the concentration of exogenous gene was 3.00 μg/mL. The positive rate of the double nucleus injection group was significantly higher than that of the single injection group (24.3% vs. 18.3%, P＜0.01). After micro-injection, the 2 cell stage embryo transplantation of 0.5 d, 1.5 and 2.5 d, to the pseudopregnant females through the fallopian tube. The birth rates were 68.6%, 67.6% and 40.1%, respectively, and the positive rate were 17.4%, 22.5% and 10.1% respectively. In comparison, the birth rate had no difference in 0.5 d and 1.5 d surrogate mothers group, but the transgenic positive rate (22.5%) in the 1.5 d group was significantly higher than that in the other groups. The results indicated that the fine as much as possible of the injection needle; 2~4 μg/mL exogenous gene concentration; double-pronuclear microinjection; 1.5 days pseudopregnant were the key to obtaining high efficiency of transgenic mice. This study may provide some useful reference for the research of transgenic animal technology.
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