原代胚胎干细胞小鼠（F0-ESC mouse）的培育及其方法学研究

发布时间：2018-01-19 03:13

本文关键词： Velocimouse技术基因修饰小鼠胚胎干细胞诱导性多能干细胞　出处：《福建医科大学》2012年硕士论文　论文类型：学位论文

【摘要】：目的利用Velocimouse技术原代（F0）培育完全来源于胚胎干细胞的小鼠（F0-ESC小鼠），分析探讨该技术的相应技术细节，以便为胚胎干细胞、诱导多能干细胞的鉴定和基因修饰小鼠制备等生命科学研究提供更为便捷、有效的技术平台。方法通过显微注射将10～15个G1-ESCs注入到野生型ICR小鼠的8细胞胚胎，转至KSOM+AA培养液滴中体外培养48h后置荧光显微镜下观察，计数囊胚率、胚胎干细胞嵌合体囊胚率和胚胎干细胞纯合子囊胚率。部分显微注射后的8-细胞胚胎置KSOM+AA培养液滴中体外培养2h后移植至假孕0.5d的野生型ICR雌鼠的输卵管内或者培养过夜后移植至假孕2.5d的野生型ICR雌鼠的子宫内。产出小鼠后蓝光激发下观察，全身均为绿色荧光且毛色为黑色的初步判断为ESC小鼠。将ESC小鼠与野生型ICR小鼠交配，根据其后代（F1）荧光观察及毛色判定ESC小鼠是否为生殖腺传递。解剖ESC小鼠，，观察各器官荧光状况判断其全身器官是否均为胚胎干细胞细胞发育而来最终确定其是否为F0-ESC小鼠。结果（1）显微注射后体外培养至囊胚，荧光显微镜检查表明18.54%的囊胚内细胞团完全由G1-ESC形成；（2）培育出3只原代含有G1-ESC的小鼠，其中1只为F0-ESC小鼠（雄性），2只嵌合体小鼠；（3）将F0-ESC小鼠与野生型ICR雌鼠交配证实其为生殖腺传递；（4）F0-ESC小鼠各器官取材荧光观察发现各器官均有荧光证实其为全身均为ES细胞发育而来的F0-ESC小鼠。结论Velocimouse技术能有效的获得原代胚胎干细胞小鼠，为胚胎干细胞、诱导多能干细胞的鉴定和基因修饰小鼠制备等生命科学研究提供了更为便捷、有效的方法。本实验室建立的G1-ESC为具有可靠多能性的优良胚胎干细胞株。
[Abstract]:The purpose of the use of Velocimouse Technology (F0) to foster a complete primary embryonic stem cells derived from mice (F0-ESC mice), analysis the technical details of the technology, so that the embryo stem cells, induced pluripotent stem cells and identification of gene modified mice, preparation of life science research and provide a more convenient and effective technology platform.
Methods by microinjection of 10 ~ 15 G1-ESCs into the 8 cell embryos of wild-type ICR mice, to KSOM+AA cultured in vitro under fluorescence microscope 48h post droplet, counting the blastocyst rate of embryonic stem cells, chimeric blastocyst rate and blastocyst rate. Embryonic stem cells homozygous for 8- cell embryos after microinjection of the part KSOM+AA cultured in 2h after transplantation to drop 0.5d pseudopregnant wild type ICR female rats in the oviduct or uterine transplantation to cultivation of wild type ICR 2.5D pseudopregnant female rats after overnight. After mice were observed under blue light excitation output, the whole body are green fluorescent color and black ESC preliminary judgment mice. ESC mice and wild-type ICR mice mated according to their offspring (F1) fluorescence observation and color to determine whether transmission ESC mouse gonads. Anatomy of ESC mice, observe the fluorescence in the systemic organ organ judgment Whether or not the embryonic stem cell cells are developed to determine whether they are F0-ESC mice.
Results (1) after microinjection and embryos cultured in vitro, fluorescence microscopy showed that 18.54% blastocyst inner cell mass was formed by G1-ESC; (2) developed 3 primary containing G1-ESC mice, 1 of which only F0-ESC mice (male), 2 chimeric mice; (3) F0-ESC mice and wild type ICR female mating was confirmed germline transmission; (4) the organs of F0-ESC mice were observed by fluorescence fluorescence confirmed that the organs of the body are developed to ES cells in F0-ESC mice.
Conclusion Velocimouse can effectively obtain primary mouse embryonic stem cells, embryonic stem cell, induced pluripotent stem cells and identification of gene modified mice, preparation of life science research provides a more convenient and effective method. The laboratory established G1-ESC has excellent reliability of embryonic stem cell lines.

【学位授予单位】：福建医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R329

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