Quantitation Of Mir-505Expression And Observation Of Related Physiological Traits In Mir-505Transgenic Mice

Puberty, often influenced by both enviromental and genetic factors, is a complicated biologic process involving sexual maturation and gaining ability of reproduction. Onset of puberty is triggered by reactivation of the hypothalamie-pituitary-gonadal axis (HPGA) and modulated by both genetic and environmental factors. Generally, when human growth and development get to a certain period, as the hypothalamus-pituitary-gonadal axis (HPGA) mature, puberty development start, but the exact molecular mechanism is unclear.In recent years, many people use mice to study the mechanism of sexual development. Beginning in2004, the laboratory, from more than400kinds of inbred strains of mice strains, screened two sexual development startup time there are significant differences, and mitochondrial genome-wide background of inbred strains of mice known C3H/HeJ and C57BL/6J as a parent to produce the F2generation hybrids, STR genome-wide scans and genetic analysis showed that in the6th, the13th and the X chromosome has been associated with the female vulva opening time section, where-2.5cM X chromosome (DXMit103-DXMit119, LOD=3.86) for QTL segment was significantly associated with the first discovered genetic effect is dominant C57BL/6J C3H/HeJ recessive. To construct the initial positioning of the mouse X chromosome segment QTL several specific sections of similar lines, the use of STR and SNP genotyping beacon combined with phenotypic identification, N7-generation pedigrees in sexual development in the mouse QTL region segment reduced to the range between the X chromosome1cM rs13483770-rs29055848, and ultimately the candidate gene is located in the miR-505.MiR-505is endogenous small molecule non-coding RNA, the miR-505primary precursor is only90bp, mature miR-505includes miR-505-3P and miR-505-5P. It can be expressed in the post-transcriptional level negative regulator of the target mRNA. Under normal conditions, the expression of miR-505-5P is higher than that of miR-505-3P.To in-depth study the impact of miR-505in sexual development, we constructed transgenic mice of whole cDNA.This study is based on stem-loop real-time quantitative RT-PCR to detect the mature miRNA; and quantify microRNA precursors using absolute quantification method. Improved stem-loop qRT-PCR, analyze samples using the method of relative quantification. To detect microRNA precursors, absolute quantitative analysis methods were used to detect the initial number of copies of the precursor. Using this technique to detect miR-505in transgenic mice and control lines C57BL/6J in four different age at5,15,25,35, and hypothalamus, pituitary, ovary, muscle, adipose five different tissue, the results show:in different days and different tissues, the expression of pri-miR-505are very low, only a few dozen copies; in5and15days,the initial copy number of pre-miR-505in transgenic mice is higher than that in C57BL/6J about10times. For mature miRNA, addition Hypothalamus, the relative expression levels of miR-505-5P are substantially greater than1in pituitary, ovary, muscle and adipose tissue, especially in ovarian tissue and fat, in the age of5and15, the relative expression level is as high as about4. However, miR-505-3P in the hypothalamus, pituitary, ovary tissue located sexual development axis, the relative expression levels are basically around1.Meanwhile, the study identified miR-505transgenic mice and inbred strain C57BL/6J phenotypes, including:vaginal opening, horny and growth curve, found no significant differences.In summary, this study successfully constructed economical.practical sensitive and efficient scheme to detect microRNA precursors and mature microRNA. Using this technique at different ages, in different tissues of transgenic mice and wild-type mice show that miR-505in transgenic mice is significantly increased transcript levels, but at the level of gene expression in sexual development related hypothalamic-pituitary-gonadal axis did not show it, show that the construction of transgenic mice was successful. At the same time, by comparing the expression of miR-505-3P and miR-505-5P of the difference in the amount of transgenic mice, indicating that associated with sexual development may be miR-505-3P in play, which requires further proved. Through this study, follow-up on the sexual development of experimental and theoretical work has laid a foundation.