Factors Affecting Production Efficiency of Bacterial Artificial Chromosome Transgenic Mice

Abstract

ObjectiveBy comparing some key factors in generating bacterial artificial chromosome (BAC) transgenic mice, such as the background of strains, the concentration of the BAC DNA used for injection, the size of the BAC DNA, and the storage time of the BAC DNA injection, to optimize the generation of BAC transgenic mice.MethodsThe same procedure of BAC DNA preparation was used with two mouse strains, i.e. C57BL/6J and FVB/N to produce embryos; four concentrations of DNA for injection (0.75, 1.0, 1.5, and 2.0 ng/µL, respectively) were utilized to microinject into the embryos. The size of BAC DNA fragment and the time from BAC preparation to injection were recorded, and the birth rate and positive rate of transgenic mice were compared.ResultsThe positive rate of transgenic mice with FVB/N background was higher than that of C57BL/6J background (P 1.5 ng/µL>1.0 ng/µL>2.0 ng/µL. When the injection concentration was 1.5 ng/µL, the positive rates transgenic mice in C57BL/6J and FVB/N newborns were the highest, while the positive rate was 0 in C57BL/6J newborns when the injection concentration was 0.75 ng/µL and 2.0 ng/µL. When different concentrations of BAC DNA were adopted, the positive rate of transgenic mice in FVB/N newborns from high to low was 1.5 ng/µL>1.0 ng/µL>2.0 ng/µL>0.75 ng/µL. When the BAC fragment size was 197 kb, the mice birth rate was highest at (22.49±9.41)%, and when the fragment size was 99 kb, the birth rate was lowest at (13.61±15.65)%. When the BAC fragment was 197 kb, the transgenic mice had the highest positive rate of (13.56±12.88)%, extremely significantly higher than that for 114 kb (P < 0.01) and significantly higher than that for 99 kb (P < 0.05). The storage time of BAC injection was not linearly associated with the positive rate of transgenic mice.ConclusionCompared with C57BL/6J, mice with FVB/N background appear more suitable for the generation of BAC transgenic mice. When the concentration of BAC DAN injection is 1.5 ng/µL, the positive rate of transgenic mice is the highest. BAC fragment size is not a constraining factor for positive transgenic mice. The highest efficiency is expected when the BAC DNA is injected within one week after preparation.

Keywords

• bacterial artificial chromosome
• transgenic mice
• embryo microinjection