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Zinc Finger Nucleases

Zinc Finger Nucleases. Derivation and Characterization of Rat Embryonic Stem Cells. Characteristics of Embryonic Stem Cells. Establishment of pluripotent embryonic cell (like) lines. Mouse ＊ , Human Monkey, Rat, Pig, Sheep…. RAT.

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Zinc Finger Nucleases

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  1. Zinc Finger Nucleases

  2. Derivation and Characterization of Rat Embryonic Stem Cells

  3. Characteristics of Embryonic Stem Cells

  4. Establishment of pluripotent embryonic cell (like) lines Mouse＊, Human Monkey, Rat, Pig, Sheep…

  5. RAT • Model for biomedical research, physiology, toxicology studies… A bigger mouse?

  6. 3I medium Ying et al, Nature ,2008, 453(7194):519-523 CHIR99021: GSK3 inhibitor PD184352: MEK1/2 SU5402: FGFR

  7. Method

  8. Fig. 1 Culture of blastocysts in different medium A: LIF+Serum; B: N2B27; C: N2B27+3I

  9. Fig. 2 Derivation and maintain of rat ES cells from rat blastocyst

  10. Fig. 3 Immunostaining for pluripotency marker in rat ES cells

  11. Fig. 4 AP staining and pluripotency gene expression in rat ES cells

  12. Fig. 5 Immunofluorescence staining for cell surface marker

  13. In vitro differentiation

  14. Fig.7 In vitro differentiation of rat ES cells Rat ES cells in MEF medium

  15. MEF medium+CDM day4 MEF medium Fig.7 In vitro differentiation of rat ES cells

  16. Fig.7 In vitro differentiation of rat ES cells MEF medium+CDM day 8

  17. Fig.8 RT-PCR analysis of gene expression in rat ES cells and EBs

  18. Fig.8 Immunostaing confirming in vitro differentiation

  19. Fig.9 X inactivation occurs during differentiation of famale rat ES cells

  20. Production of Chimera

  21. A DAc2 B DAc8 Fig.10 Karyotype analysis of the rat ES cells A: G banding; B: Gender verification by PCR

  22. Ovary Uterus Fig.11 Exposure of uterus for blastocysts tansferring

  23. Fig.12 Chimeric rat produced by injection of DAc2 into SD blastocysts

  24. Fig.14 Chimeric rat and germline produced by injection of DAc2 into F344 blastocysts

  25. A: 153bp; a: 134bp Fig.15 Genotyping analysis of germline rats and its pups D: SSLP (Simple Sequence Length Polymorphism)

  26. LIF/STAT3 signaling in rat ES cells

  27. Fig. 16 Response of rat ES cells to LIF stimulation

  28. Fig. 21 Bisulfite sequencing of DNA methylation of the Oct4 and Nanog promoter regions CU

  29. Cell, 2008

  30. Nature, 2010

  31. Thank You!

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