Isolation And Detection Of Hybrid ES Cells From EGFP Transgenic Mouse

This study successfully isolated embryonic stem cells from hybrid blastocysts which were the results of breeding male EGFP transgenic mouse with female KM (Kunming) mouse. A series of tests were detected: morphological observation, karyotype analysis, alkaline phosphatase staining, staining of SSEA-1 and OCT-4, differentiation of embryoid bodies and teratoma. Then, chimeras were produced to study in-vivo differentiation of ES cell. Resultant male chimeras were bred with female KM mouse to examine germ-line transmission ability. Specific procedures were divided into two parts.1. Isolation and detection of ES cell lines from hybrid blastocysts(1) Superovulated KM female mouse (more than 6 weeks) was bred with male EGFP transgenic mouse (8 weeks) which was fed in single cage. Embolus was checked for the fertilized mouse next day and it was 0.5 days of fertilization. After 3.5 days the pregnant mice were killed to collect embryos through flushing uterus with M2 medium. Twenty hybrid embryos were harvested, which had fifteen blastocysts and five morulas. Seven blastocysts were chosen to place directly on treated feeder cell layer cultured in ESM (embryonic stem medium). After 1-2 days of culture which were attached to the surface of tissue culture dish, the embryos hatched from zona pellucida. Five blastocysts hatched successfully and developed into ICM-derived clump.(2) Import mitomycin was mostly used to treat fibroblast as feeder layer. Although its effect was obvious, price was high and the process of purchase was time-consuming and laborious. Homemade Mitomycin had price edge, but its effect was unknown and few people attempted to prove it. We used homemade mitomycin to have a bold try. Mouse embryonic fibroblasts (MEF) treated by homemade mitomycin were used as feeder layer. Finally mouse embryonic stem cells were successfully isolated.(3) One of ES cell lines was detected by conventional ES cell tests. The results were as follows:Morphological observation: cells formed tightly packed colonies and the shape of it liked nest. The edge of cells was hard to define. Single cell was small and round with big nucleus and small cytoplasm after digestion treatment. Fluorescence microscopy showed that ES cell clones have a strong green fluorescence.Karyotype Analysis: ES cells had a normal diploid karyotype (a total of 40 chromosomes).Alkaline phosphatase (AKP) staining: ES cell clones were stained dark blue, showing alkaline phosphatase activity.SSEA-1 and OCT-4 staining: positive.Embryoid body and teratoma: EGFP-ES cell could differentiate in vitro, which showed good differentiation potential and resultant embryoid bodies had a strong green fluorescence under fluorescent microscope. There were all kinds of tissue cells in the teratomas acquired from in-vitro differentiation, such as, neuro epithelium (ectoderm), skeletal muscle (mesoderm), respiratory epithelium (endoderm). This further proved the isolated mouse ES cells could differentiate into three germ layers of tissue types.2. The study on in-vivo differentiation of ES cell line from hybrid blastocysts' production and detection of chimera.(1) Flushing uterus, 3.5 days mouse blastocysts were collected as recipient embryos. Single cell suspension of ES cells (10-20 passage) was prepared as donor cells. Then ES cells were injected into blastocyst cavity, 10-15 ES cells/ one blastocyst.(2) Blastocyst cavity vanished after injection. After 1-3 hours incubation, 84 blastocysts which reappeared cavities were selected to implant into eight pseudo-pregnant uteruses. Pregnant duration was 19.5 days. The repeat of experiment was 8 times. Five pregnant mice successfully delivered 13 fetuses, nine of which were chimeric mouse. The numbers of high, medium and low degree chimeric mice were five, two and two respectively. The coat color of high-degree chimeric mouse was basically black or gray. Medium-degree chimeric mouse was between high-degree and low-degree chimeric mouse. Low-degree chimeric mouse had a slice of black or gray fur.(3) Germ-line transmission test: Five high-degree chimeric male mice were chosen to breed with five female KM mice. Two groups of five had 13 offspring respectively, of which had eight other color phenotype respectively. Another three groups did not deliver any fetuses except one group with all white offspring. The hybrid (F1) color phenotype got from breeding KM female mouse with chimeric male one showed that the strain of ES cells had germ-line transmission ability. The Karyotype of hybrid ES cell was XY because of appearance of male offspring from ES cell.(4) Flow cytometry test: Two high-degree and one low-degree chimeric mice were selected to do the test. Their hearts, spleens, kidneys, and marrows were digested into single-cell suspension. Flow cytometry results showed that the chimeric rates of EGFP-ES heart, spleen, kidney, marrow were 52.02%, 77.96%, 84.06% and 42.49% respectively.