The chimeric mice derived from umbilical cord blood stem cells of EGFP-transgenic mouse

Abstract

Objective: The chimeric mice were prepared by microinjection of blastocyst cavity using umbilical cord blood stem cells (UCBSCs) of Enhanced Green Fluorescent Protein (EGFP)-transgenic mouse, which was expected to provide a theoretical and experimental basis for the study of in-vivo differentiation of adult stem cells.

Methods: Mouse UCBSCs expressing green fluorescence was microinjected into blastocyst cavity and several blastocysts were transferred into uterus of pseudo pregnant mouse. First of all, new-born candidate chimeric mice were observed through feather color. Secondly, the genomic DNA and total RNA were extracted to analyze chimeric rate in several tissues. Finally, flow cytometry was used to detect percentage of green fluorescent cells mice in several tissues. 

Results: The UCBSCs expressing green fluorescent protein were successfully isolated. After flow cytometry analysis, the proportion of cells expressing green fluorescence was 80.25%. Through microinjection and embryo transfer, we got five white new-born mice and no chimeric feather color was observed. The analyses of PCR and RT-PCR were carried out to detect EGFP gene using six tissues including heart muscle, liver, lung, skin, leg muscle and adipose tissue. The results showed that the leg muscle and adipose tissue of two mice were positive and the other tissues and six tissues of the other 3 mice were all negative. The leg muscle and adipose tissue of two positive mice were digested into single-cells suspension and were carried out flow cytometry analysis. The results showed that the average chimeric rates of leg muscle and adipose tissue of two positive mice were 9.87% and 5.78%, respectively.

Conclusions: The results demonstrated that adult UCBSCs could differentiate into leg muscle and adipose tissue in vivo.