Development of Nuclear Transfer and Embryonic Stem Cell Technology in Mouse

Abstract

The objectives of this study were to compare the efficiency of embryonic stem cells (ESCs) derived from fertilized blastocysts of different mouse strains (EXP. 1), to compare the efficiency of ESCs derivation and characterization of ESCs derived from cloned and fertilized blastocysts (EXP. 2) and to compare the potential of in vitro differentiation of ESCs derived from cloned and fertilized blastocysts (EXP. 3). EXP 1. The strains of mouse used in this experiment were C57BL/6 (inbred), B6D2F1 (hybrid) and CD1(outbred). Firstly, 40-44 expanded blastocysts developed in vivo were collected and subjected to ICM number evaluation. Secondly, Pou5fl positive cells were detected in 10-14 expanded blastocyts. Thirdly, Efficiency of ESC derivation from different mouse strains was compared. Expanded blastocysts of inbred showed no significant difference in ICM number compared to hybrid but significantly higher than outbred. The Pou5f1 positive cells were detected in all blastocysts of three examined strains. No significant difference of efficiency in ESCs derived from expanded blastocysts of inbred (16.7%) and hybrid strain (31.6%) but no ESC line was obtained from the outbred strain. Finally in additional experiments, delayed blastocysts of outbred strain were used to evaluate ICM number, detect Pou5fl and establish ESC line. Delayed blastocyst of outbred showed significantly higher ICM number than expanded blastocysts of the same strain. Pou5fl was detected in delayed blastocysts and outbred ESC lines can be derived from delayed blastocysts. This finding indicated that efficiency of ESC derived from expanded blastocysts of inbred and hybrid strain was not different regarded to no difference of ICM number. Outbred ESC lines can be derived from delayed blastocysts but not expanded blastocysts. EXP 2. Cloned and fertilized blastocysts were subjected to Pou5fl positive cells detection. Efficiency of ESC derived from cloned and fertilized blastocysts were compared. Pluripotency of ESCs derived from cloned and fertilized blastocysts were detected by specific markers and gene expression of ESCs. Pou5fl positive cells were detected in cloned (10/10) and fertilized blastocysts (10/10). Efficiency of ESCs derived from cloned [15.4%; 4/26)] was significantly lower than fertilized blastocysts [62.5%; 15/24]. ESCs derived from cloned displayed specific markers, gene expression and normal chromosome number similar to those derived from fertilized blastocysts. This study indicated that although Pou5fl positive cells were detected in cloned as in fertilized blastocysts, the efficiency of ESC derivation of the former was inferior. By detecting specific markers, gene expression and chromosome number, ESCs derived from cloned and fertilized blastocysts display similarity. EXP 3. Nuclear transfer-derived ESCs (NT-ESCs) and fertilization-derived ESCs (F-ESCs) derived EBs were produced in hanging drops, cultured in suspension and in vitro differentiation potential were compared. Mean of diameter of EBs were measured every 3-4 day up to Day 26. Histomorphology and gene expression were investigated in 1, 2, 3, 4 and 5-week-old EBs. EBs formation efficiency of NT-ESCs did not significantly differ from F-ESCs (95% vs 96.8% respectively). NT-ESCs derived EBs showed significantly greater diameter than F-ESCs derived EBs in Day 7 and 10. Primitive neural tubes were found in NT-ESCs and F-ESCs derived EBs at Day 14. Expression pattern of AFP in NT-ESCs and F-ESCs derived EBs was different. This study showed some differences between NT-ESCs and F-ESCs derived EBs which will be useful for further study of comparative characteristics of NT-ESCs and F-ESCs.