动物造模 z-bio 2022-08-19 448

　　Objective To compare several key factors in the development of bacterial artificial chromosome transgenic mice, such as the genetic background of experimental mice, BAC plasmid injection concentration, BAC plasmid fragment size and BAC injection storage time, and optimize the development technology of BAC transgenic mice.

　　Methods Using the same BAC DNA preparation method, two experimental mice, C57BL/6J (B6) and FVB/N, were selected, and BAC injections with mass concentrations of 0.75, 1.0, 1.5 and 2.0 ng/µL were used for embryo microscopy. After injection, the size of different BAC DNA fragments and the length of time from the preparation of BAC vector DNA to before injection were analyzed, and the birth rate of mice and the positive rate of transgenic mice were compared.

　　Results The positive rate of transgenic mice with FVB/N as background was higher than that with C57BL/6J (P<0.05). the="" birth="" rates="" of="" 6j="" and="" n="" pups="" corresponding="" to="" injection="" mass="" concentrations="" four="" bac="" plasmids="" were="" ranked="" from="" high="" low:="" 0.75="" l="" group="">1.5 ng/µL group>1.0 ng/µL group>2.0 ng/µL group. When 4 BAC plasmid concentrations were injected, the positive rates of C57BL/6J transgenic mice were ranked from high to low as 1.5 ng/µL>1.0 ng/µL>2.0 ng/µL=0.75 ng/µL, FVB/N transgenic mice The order of positive rate from high to low was 1.5 ng/µL>1.0 ng/µL>2.0 ng/µL>0.75 ng/µL. When the size of the BAC fragment was 197 kb, the birth rate of offspring was the highest (22.49±9.41)%; when the fragment size was 99 kb, the birth rate of offspring was the lowest, (13.61±15.65)%; when the BAC fragment was 197 kb, the transgenic The highest positive rate was (13.56±12.88)% in mice, which was significantly higher than 114 kb (P＜0.01) and significantly higher than 99 kb (P＜0.05). There is no linear relationship between the storage time of BAC injection and the positive rate of transgenic mice.

　　Conclusion Compared with C57BL/6J, the FVB/N background is more suitable for the development of BAC transgenic mice. When the mass concentration of BAC injection was 1.5 ng/µL, the preparation efficiency of positive transgenic mice was the highest. BAC fragment size is not a limiting factor for positive transgenic mice. The best results were achieved when the injection was completed within 1 week of BAC vector DNA preparation.