演讲嘉宾

李劲松


中科院生物化学与细胞生物学研究所研究员,课题组长,博士生导师

会议报告摘要:生殖干细胞介导的基因编辑

哺乳动物生殖干细胞和CRISPR-Cas9技术的建立为生命科学研究提供了新的工具。已有的生殖干细胞有两类,一是能代替精子使用的孤雄单倍体胚胎干细胞,二是精原干细胞。CRISPR-Cas9技术由于其简单高效的特点很快在生命科学研究中得到了广泛的应用。2012年,我们建立了只携带精子来源遗传物质的小鼠孤雄单倍体胚胎干细胞,并证明这一细胞能代替精子在注入卵母细胞后能支持胚胎发育产生健康的半克隆小鼠,即半克隆技术。然而,单倍体细胞的“受精”能力随着细胞的传代逐渐丢失,特别是经过基因编辑后,这些细胞再注入卵子中很难获得健康半克隆小鼠。
最近,我们通过将调控雄性印记基因H19和Gtl2表达的H19-DMR和IG-DMR敲除后获得了能稳定产生半克隆小鼠的“人造精子”。与CRISPR-Cas9技术结合,“人造精子”介导的半克隆技术可以实现:(1)一步获得携带多基因突变的杂合小鼠模型,用于模拟人类多基因介导的复杂疾病;(2)一步获得多基因同时敲入的小鼠模型;(3)一步获得针对不同基因的突变小鼠,实现小鼠个体水平的遗传筛选,便于从大量候选基因中快速筛选出重要基因进行深入研究。
精原干细胞已经在小鼠等物种中建系,并能在体外长期稳定传代并具有产生配子的能力。与CRISPR-Cas9技术结合,精原干细胞可以用于:(1)治疗雄性遗传疾病,使得后代完全不携带遗传缺陷;(2)开展减数分裂与精子发生的研究。综上,生殖干细胞与CRISPR-Cas9技术的结合将极大促进生命科学的研究。


研究方向

细胞重编程与胚胎发育


研究经历

1989-1993年 江西农业大学 畜牧兽医系 学士
1993-1996年 扬州大学 畜牧兽医学院 硕士
2002年 中国科学院动物研究所 博士
2002-2007年 美国 洛克菲勒大学 博士后
2007年—今 中科院上海生科院 生物化学与细胞生物学研究所 研究院组长


研究工作

细胞重编程(cellular reprogramming)指的是细胞在特定的条件下被逆转恢复到全能性状态或多能性状态,或者形成多能干细胞系,或者进一步发育成一个新的个体的过程。核移植与诱导多能干细胞(induced pluripotent stem cell, iPSC)技术是两种主要的重编程方法。然而这两种技术都还很不成熟,存在效率很低、重编程细胞发育能力差、重编程的机制不清楚等问题。

2012年,研究组建立了来自小鼠精子的孤雄单倍体胚胎干细胞,并证明这些细胞能够代替精子使卵母细胞“受精”产生半克隆小鼠(称为“半克隆”技术)。然而,半克隆技术不成熟,半克隆小鼠出生率低(4%左右),而且有一半的半克隆小鼠存在发育缺陷问题。2015年,研究组获得了能稳定高效产生半克隆小鼠的“类精子细胞”的单倍体干细胞(出生效率达到20%左右),并证明这些细胞能携带CRISPR-Cas9文库一步获得大量携带不同突变的小鼠,为实现小鼠个体水平的遗传筛选奠定了基础:
1.完善核移植及iPSC技术诱导细胞重编程的方法,提高重编程的效率;
2. 探讨重编程细胞发育缺陷相关的分子机制,提高重编程细胞的质量;
3. 探讨半克隆小鼠发育缺陷的相关分子机制,揭示与胚胎发育相关的重要机制;
4. 结合“类精子细胞”的单倍体干细胞与CRISPR-Cas9技术,开展新的基因编辑应用研究;
5. 开展CRISPR-Cas9技术介导的基因治疗研究。


近期发表论文

  1. Zhong C#, Yin Q#, Xie Z#, Bai M#, Dong R#, Tang W, Xing Y, Zhang H, Yang S, Chen L-L, Bartolomei MS, Ferguson-Smith A, Li D, Yang L*, Wu Y*&Li Jinsong*. CRISPR-Cas9-mediated genetic screening in mice with haploid embryonic stem cells carrying a guide RNA library. Cell Stem Cell2015, 17, 221-232.
  2. Bosley KS, Botchan M, Bredenoord AL, Carroll D, Charo RA, Charpentier E, Cohen R, Corn J, Doudna J, Feng G, Greely HT, Isasi R, Ji W, Kim J, Knoppers B, Lanphier E, Li Jinsong, Lovell-Badge R, Martin GS, Moreno J, Naldini L, Pera M, Perry A, Venter JC, Zhang F & Zhou Q. CRISPR germline engineering-the community speaks. Nature Biotechnology 2015, 33, 5, 478-486. (Feature)
  3. Liu P, Dou X, Liu C, Wang L, Xing C, Peng G, Chen J, Yu F, Qiao Y, Song L, Wu Y, Yue C, Li Jinsong, Han J, Tang K & Jing N. Histone deacetylation promotes mouse neural induction by restricting Nodal-dependent mesendoderm fate. Nature Communications 2015, 6: 6830.
  4. Qin Y, Qin J, Zhou C, Li Jinsong& Gao W. Generation of embryonic stem cells from mouse adipose-tissue derived cells via somatic cell nuclear transfer. Cell Cycle 2015, 14: 1282-1290.
  5. Zhu W, Yao X, Liang Y, Liang D, Song L, Jing N, Li Jinsong& Wang G*. Mediator Med23 deficiency enhances neural differentiation of murine embryonic stem cells through modulating BMP signaling. Development 2015, 142: 465-476. Epub Jan 6. pii: dev.112946.
  6. Wu Y#, Zhou H#, Fan X#, Zhang Y#, Zhang M#, Wang Y, Xie Z, Bai M, Yin Q, Liang D, Tang W, Liao J, Zhou C, Liu W, Zhu P, Guo H, Pan H, Wu C, Shi H, Wu L*, Tang F* &Li Jinsong*. Correction of a genetic disease by CRISPR-Cas9-mediated gene editing in mouse spermatogonial stem cells. Cell Res 2015, 25: 67-79. Epub 2014 Dec 5.
  7. LianX, Xu J,Li Jinsong& Chien K. Next-generation models of human cardiogenesis via genome editing. Cold Spring Harb Perspect Med 2014 Sep 18;4(12). pii: a013920. doi: 10.1101/cshperspect.a013920.
  8. Guo F#, Li X#, Liang D#, Li T#, Zhu P, Guo H, Wu X, Wen L, Gu T, Hu B, Walsh CP, Li Jinsong*, Tang F* & Xu G*. Active and passive demethylation of male and female pronuclear DNA in the mammalian zygote. Cell Stem Cell 2014, 15: 447-458.[Epub ahead of print].
  9. Zhang M, Zhou H, Zheng C, Xiao J, Zuo E, Liu W, Xie D, Shi Y, Wu C, Wang H, Li D & Li Jinsong. The roles of testicular c-kit positive cells in de novo morphogenesis of testis. Sci Rep 2014, 4: 5936.
  10. Zhu Q, Song L, Peng G, Sun N, Chen J, Zhang T, Sheng N, Tang W, Qian C, Qiao Y, Tang K, Han JD, Li Jinsong& Jing N. The transcription factor Pou3f1 promotes neural fate commitment via activation of neural lineage genes and inhibition of external signaling pathways. Elife 2014, Jun 14:e02224. doi: 10.7554/eLife.02224. [Epub ahead of print].
  11. Zhao B, Yang D, Jiang J, Li Jinsong, Fan S, Huang M, Fan Y, Jin Y & Jin Y. Genome-wide mapping of miRNAs expressed in embryonic stem cells and pluripotent stem cells generated by different reprogramming strategies. BMC Genomic 2014, 15: 488.
  12. Yu Y, Liang D, Tian Q, Chen X, Jiang B, Chou BK, Hu P Cheng L, Gao P, Li Jinsong& Wang G. Stimulation of somatic cell reprogramming by Eras-Akt-Foxo1 signaling axis. Stem Cells 2014,32(2): 349-363. doi: 10.1002/stem.1447. [Epub ahead of print].
  13. Qin Y, Zhou P, Zhou C, Li Jinsong& Gao W. The adipose-derived lineage-negative cells are enriched mesenchymal stem cells and promote limb ischemia recovery in mice. Stem Cells Dev 2014, 23: 363-371.
  14. Wu Y#, Liang D#, Wang Y, Bai M, Tang W, Bao S, Yan Z, Li D &Li Jinsong*. Correction of a genetic disease in mouse via use of CRISPR-Cas9. Cell Stem Cell 2013, 13: 659-662.
  15. Yang H#, Liu Z#, Ma Y#, Zhong C, Yin Q, Zhou C, Shi L, Cai Y, Zhao H, Wang H, Tang F, Wang Y, Zhang C, Liu X, Lai D, Jin Y*, Sun Q* &Li Jinsong*. Generation of haploid embryonic stem cells from Macaca fascicularis monkey parthenotes. Cell Res 2013, 23: 1187-1200.
  16. Qin Y#, Lin J#, Zhou C#, Yin Q, Xie Z, Zhang X, Liu X, Gao W &Li Jinsong*. Mice cloned from white adipose tissue-derived cells. J Mol Cell Biol 2013, 5: 348-350.
  17. Jiang J#, Lv W#, Ye X, Wang L, Zhang M, Yang H, Okuka M, Zhou C, Zhang X, Liu L* & Li Jinsong*. Zscan4 promotes genomic stability during reprogramming and dramatically improves the quality of iPS cells as demonstrated by tetraploid complementation. Cell Res2013, 23: 92-106.
  18. Zhao S#, Gou LT#, Zhang M#, Zu LD, Hua MM, Hua Y, Shi HJ, Li Y, Li Jinsong, Li DS, Wang ED* & Liu M. piRNA-triggered MIWI ubiquitination and removal by APC/C in late spermatogenesis. Dev Cell 2013, 24: 13-25.
  19. Yang H#, Shi L#, Wang B#, Liang D, Zhong C, Liu W, Nie Y, Liu J, Zhao J, Gao X, Li D, Xu G-L*&Li Jinsong*. Generation of genetically modified mice by oocyte injection of androgenetic haploid embryonic stem cells. Cell 2012, 149(3): 605-617.
  20. Shi L, Yang H &Li Jinsong*. Haploid embryonic stem cells: an idea tool for mammalian genetic analyses. Protein & Cell 2012, 3: 806-810. (Invited review)
  21. Ma Y, Gu J, Li C, Wei X, Tang F, Shi G, Jiang J, Kuang Y, Li Jinsong, Wang Z, Xie X & Jin Y. Human foreskin fibroblast produces interleukin-6 to support derivation and self-renewal of mouse embryonic stem cells.Stem Cell Res Ther 2012, 3: 29.
  22. Gu TP#, Guo F#, Yang H#, Wu HP, Xu GF, Liu W, Xie ZG, Shi L, He X, Jin SG, Iqbal K, Shi YG, Deng Z, Szabo PE, Pfeifer GP, Li Jinsong* & Xu GL*. The role of Tet3 DNA dioxygenase in epigenetic reprogramming by oocytes. Nature 2011, 477, 606-610.
  23. Jiang J#, Ding G#, Lin J#, Zhang M, Shi L, Lv W, Yang H, Xiao H, Pei G, Li Y, Wu J* &Li Jinsong*. Different developmental potential of pluripotent stem cells generated by different reprogramming strategies. J Mol Cell Biol2011, 3: 197-199.
  24. Lin J, Shi L, Zhang M, Yang H, Qin Y, Zhang J, Gong D, Zhang X, Li D &Li Jinsong*. Defects in trophoblast cell lineage account for the impaired in vivo development of cloned embryos generated by somatic nuclear transfer. Cell Stem Cell2011, 8: 371-375.
  25. Yang H#, Shi L#, Chen D &Li Jinsong*. Mice generated after round spermatid injection into haploid two-cell blastomeres. Cell Res2011, 21: 854-858.
  26. Li X, Zhu L, Yang A, Lin J, Tang F, Jin S, Wei Z, Li Jinsong&Jin Y. Calcineurin-NFAT signaling critically regulates early lineage specification in mouse embryonic stem cells and embryos. Cell Stem Cell 2011 8: 46-58.
  27. Wang Y, Chen J, Hu J, Wei X, Qin D, Gao J, Zhang L, Jiang J, Li Jinsong, Liu J, Lai K, Kuang X, Zhang J, Pei D & Xu G. Reprogramming of mouse and human somatic cells by high-performance engineered factors.EMBO Rep 2011, 12: 373-378.
  28. Yang H, Shi L, Zhang S, Ling J, Jiang J&Li Jinsong*. High-efficiency somatic reprogramming induced by intact MII oocytes.Cell Res 2010, 20, 1034-42.
  29. Li L, Sun L, Gao F, Jiang J, Li C, Gu J, Wei Z, Yang A, Lu R, Ma Y, Tang F, Kwon SW, Zhao Y, Li Jinsong& Jin Y. Stk40 links the pluripotency factor Oct4 to the Erk/MAPK pathway and controls extraembryonic endoderm differentiation. Proc Natl Acad Sci USA 2010, 107(4):1402-1407.
  30. Chen T, Yuan D, Wei B, Jiang J, Kang J, Ling K, Gu Y, Li Jinsong, Xiao L& Pei G. E-cadherin-mediated cell-cell contact is critical for induced pluripotent stem cell generation. Stem Cells2010, 28: 1315-1325.
  31. Zhu Z, Wang Y, Li X, Wang Y, Xu L, Wang X, Sun T, Dong X, Chen L, Mao H, Yu Y, Li Jinsong, Chen PA&Chen CD. PHF8 is a histone H3K9me2 demethylase regulating rRNA synthesis.Cell Res2010, 20: 794-801.