2022, Vol. 26 ›› Issue (30): 4879-4883

Optimization of conditions for intrauterine electroporation transfection in neural stem cells from the subependymal region of fetal mice

Zou Mingming1, Ni Li2, Zhou Liyu2, Li Di3, Saijilafu2, Wei Shanwen2, Zhang Pengfei1   

Abstract: BACKGROUND: Neural stem cells are a group of cells that can self-proliferate and have multidirectional differentiation potential. In utero electroporation, which is the most direct and reliable method to study the development of cerebral cortex in vivo, can transduce RNA or plasmids into neural stem cells of mice to interfere with or overexpress target genes.
OBJECTIVE: To optimize the conditions of in utero electroporation by targeting neural stem cells in the subependymal region of embryonic mice and using enhanced green fluorescent protein as the reporter gene, so as to provide an effective method for studying the development of cerebral cortex.
METHODS: Adult ICR mice were mated at the ratio of one male to three female mice. The mice were caged at 8 p.m., and female mice with vaginal embolus were tested at 8 a.m. the next morning. The female mice with vaginal embolus were counted as 0.5-day pregnancy. On embryonic day 14 (E14), the pregnant mice were intraperitoneally anesthetized with ketamin (120 mg/kg) and xylazine (10 mg/kg). After shaving off the hair of the pregnant mouse’s abdomen and opening the abdominal cavity without cutting the pregnant mouse’s uterus, pEx-4-eGFP plasmid was transfected into neural stem cells of the subependymal region of fetal mice at different voltages using ECM803 electroporation transfection instrument and BTX electrode. After 3 days of transfection, the transfected mouse brain tissue was removed and frozen sections were performed to observe the expression of enhanced green fluorescent protein fluorescence on the cerebral cortex. The number of enhanced green fluorescent protein-positive cells was counted to evaluate the transfection efficiency.
RESULTS AND CONCLUSION: (1) The survival and transfection effect of mice were different at 38, 40, 42, and 45 V. The mortality of fetal mice was less at 38 V, but the transfection efficiency was lower. Mice treated at 42 V survived and the fluorescence intensity of enhanced green fluorescent protein was higher in the brain tissue treated at 42 V than in the brain treated at 40 V. However, 45 V was more lethal to mice. Therefore, 42 V was a better transformation condition. (2) Under the condition of 42 V, enhanced green fluorescent protein positive cells were more and the fluorescence was brighter after the pulse was changed from 5 to 8. (3) The improved conditions had no effect on the growth of transfected cortical cells.
Key words: uterus, electroporation, transfection, neural stem cells, voltage, impulse, transfection efficiency