Long noncoding RNAs (lncRNAs) modulate gene expression as competing endogenous RNAs (ceRNAs) via sponging microRNAs (miRNAs). However, the extent and functional consequences of ceRNAs in diverse cellular context still need to be proven. Using a doxycycline inducible expression of Yamanaka four factors to generate induced pluripotent stem cells (iPSCs) from mouse embryonic fibroblasts (MEFs), we found the miRNAs from MEFs remained highly expressed from day 0 to day 6 after doxycycline induction; unexpectedly, many genes targeted by these miRNAs were actually up-regulated; meanwhile, long intergenic noncoding RNAs (lincRNAs) and circular RNAs (circRNAs) which have complementary binding sites with the miRNAs were highly expressed, indicating lincRNAs and circRNAs (linc/circRNAs) may serve as sponges for miRNAs to block their activities during reprogramming. Intriguingly, the knockdown of the linc/circRNAs sponging the miRNAs targeting Oct4 mRNA resulted in down-regulation of exogenous Oct4 expression, decrease of reprogramming efficiency, and low-grade chimera forming iPSCs. Our results suggest that the ceRNA network plays an important role in reprogramming somatic cells to pluripotent stem cells.
Sperm-mediated gene transfer(SMGT) is a simple method for producing transgenic animals. Due to the lack of repeatability in spermatozoa binding and internalization of exogenous DNA, the efficiency of SMGT is still low. Considering this point, the present work aims to develop a method for evaluating the spermatozoa capacity of binding exogenous DNA after co-incubation with DNA. The main approach is using a Cy5-labelled DNA to trace the exogenous DNA and assess the ability of spermatozoa to take up exogenous DNA. Using this technique, we found that the percentage of spermatozoa that are binding and uptaking DNA is higher at concentration of 10 μg/mL and 100 μg/mL than 5 μg/mL, 1 μg/mL and 0 μg/mL after incubation with Cy5-DNA for 30min at 37 o C. After fertilization, the DNA fluorescence signal was also detected in zygotes in groups where spermatozoa were incubated with 10 μg/mL and 100 μg/mL of Cy5-DNA. These results showed a simple and convenient method to trace the exogenous DNA in spermatozoa and zygote when compared to conventional methods of labeling DNA during fertilization, resulting in a real-time observation of the exogenous DNA in spermatozoa and zygote.
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