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Neurochemical Research

, Volume 41, Issue 8, pp 2065–2074 | Cite as

Generation of Human Embryonic Stem Cell Line Expressing zsGreen in Cholinergic Neurons Using CRISPR/Cas9 System

  • Jing Zhou
  • Chencheng Wang
  • Kunshan Zhang
  • Yingying Wang
  • Xi Gong
  • Yanlu Wang
  • Siguang LiEmail author
  • Yuping LuoEmail author
Original Paper

Abstract

Lineage specific human embryonic stem cell (hESC) reporter cell line is a versatile tool for biological studies on real time monitoring of differentiation, physiological and biochemical features of special cell types and pathological mechanism of disease. Here we report the generation of ChAT-zsGreen reporter hESC line that express zsGreen under the control of the choline acetyltransferase (ChAT) promoter using CRISPR (Clustered Regularly Interspersed Short Palindromic Repeats)/Cas9 system. We show that the ChAT-zsGreen hESC reporter cell lines retain the features of undifferentiated hESC. After cholinergic neuronal differentiation, cholinergic neurons were clearly labeled with green fluorescence protein (zsGreen). The ChAT-zsGreen reporter hESC lines are invaluable not only for the monitoring cholinergic neuronal differentiation but also for study physiological and biochemical hallmarks of cholinergic neurons.

Keywords

Embryonic stem cell Cholinergic neurons CRISPR/Cas9 system Reporter cell line zsGreen 

Notes

Acknowledgments

This work is partially supported by the National Natural Science Foundations of China (Nos. 31571405, 31271450, 31271375 and 31171317) and Jiangxi Natural Science Foundation 20152ACB20008 and 20132BAB204033.

References

  1. 1.
    Deutsch JA (1971) The cholinergic synapse and the site of memory. Science 174:788–794CrossRefPubMedGoogle Scholar
  2. 2.
    Bartus RT, Johnson HR (1976) Short-term memory in the rhesus monkey: disruption from the anti-cholinergic scopolamine. Pharmacol Biochem Behav 5:39–46CrossRefPubMedGoogle Scholar
  3. 3.
    Drachman DA, Leavitt J (1974) Human memory and the cholinergic system. A relationship to aging? Arch Neurol 30:113–121CrossRefPubMedGoogle Scholar
  4. 4.
    Power AE, Vazdarjanova A, Mcgaugh JL (2003) Muscarinic cholinergic influences in memory consolidation. Neurobiol Learn Mem 80:178–193CrossRefPubMedGoogle Scholar
  5. 5.
    Schliebs R, Arendt T (2011) The cholinergic system in aging and neural degradation. Behav Brain Res 221:555–563CrossRefPubMedGoogle Scholar
  6. 6.
    Alzheimer’s Association (2014) 2014 Alzheimer’s disease facts and figures. Alzheimers Dement 10:e47–e92CrossRefGoogle Scholar
  7. 7.
    Mesulam M, Shaw P, Mash D, Weintraub S (2004) Cholinergic nucleus basalis tauopathy emerges early in the aging-MCI-AD continuum. Ann Neurol 55:815–828CrossRefPubMedGoogle Scholar
  8. 8.
    Geula C, Nagykery N, Nicholas A, Wu CK (2008) Cholinergic neuronal and axonal abnormalities are present early in aging and in Alzheimer disease. J Neuropathol Exp Neurol 67:309–318CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Nyakas C, Granic I, Halmy LG, Banerjee P, Luiten PG (2011) The basal forebrain cholinergic system in aging and dementia. Rescuing cholinergic neurons from neurotoxic amyloid-B42 with memantine. Behav Brain Res 221:594–603CrossRefPubMedGoogle Scholar
  10. 10.
    Szutowicz A, Bielarczyk H, Jankowska-Kulawy A, Pawełczyk T, Ronowska A (2013) Acetyl-CoA the key factor for survival or death of cholinergic neurons in course of neurodegenerative diseases. Neurochem Res 38:1523–1542CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Ovchinnikov DA, Titmarsh DM, Fortuna PR, Hidalgo A, Alharbi S, Whitworth DJ, Cooper-White JJ, Wolvetang EJ (2014) Transgenic human ES and iPS reporter cell lines for identification and selection of pluripotent stem cells in vitro. Stem Cell Res 13:251–261CrossRefPubMedGoogle Scholar
  12. 12.
    Krentz NA, Nian C, Lynn FC (2014) TALEN/CRISPR-mediated eGFP knock-in add-on at the OCT4 locus does not impact differentiation of human embryonic stem cells towards endoderm. PLoS One 9:e114275CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Skak K, Michelsen BK (1999) The TATA-less rat GAD65 promoter can be activated by Sp1 through non-consensus elements. Gene 236:231–241CrossRefPubMedGoogle Scholar
  14. 14.
    Wang P, Wang SM, Hsieh CJ, Chien CL (2006) Neural expression of alpha-internexin promoter in vitro and in vivo. J Cell Biochem 97:275–287CrossRefPubMedGoogle Scholar
  15. 15.
    Zhang GR, Li X, Cao H, Zhao H, Geller AI (2011) The vesicular glutamate transporter-1 upstream promoter and first intron each support glutamatergic-specific expression in rat postrhinal cortex. Brain Res 1377:1–12CrossRefPubMedGoogle Scholar
  16. 16.
    Cao H, Zhang GR, Wang X, Kong L, Geller AI (2008) Enhanced nigrostriatal neuron-specific, long-term expression by using neural-specific promoters in combination with targeted gene transfer by modified helper virus-free HSV-1 vector particles. BMC Neurosci 9:37CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Hao MM, Bornstein JC, Young HM (2013) Development of myenteric cholinergic neurons in ChAT-Cre;R26R-YFP mice. J Comp Neurol 521:3358–3370CrossRefPubMedGoogle Scholar
  18. 18.
    Boskovic Z, Alfonsi F, Rumballe BA, Fonseka S, Windels F, Coulson EJ (2014) The role of p75NTR in cholinergic basal forebrain structure and function. J Neurosci 34:13033–13038CrossRefPubMedGoogle Scholar
  19. 19.
    Foong JP, Tough IR, Cox HM, Bornstein JC (2014) Properties of cholinergic and non-cholinergic submucosal neurons along the mouse colon. J Physiol 592:777–793CrossRefPubMedPubMedCentralGoogle Scholar
  20. 20.
    Erickson CS, Lee SJ, Barlow-Anacker AJ, Druckenbrod NR, Epstein ML, Gosain A (2014) Appearance of cholinergic myenteric neurons during enteric nervous system development: comparison of different ChAT fluorescent mouse reporter lines. Neurogastroenterol Motil 26:874–884CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N, Hsu PD, Wu X, Jiang W, Marraffini LA, Zhang F (2013) Multiplex genome engineering using CRISPR/Cas systems. Science 339:819–823CrossRefPubMedPubMedCentralGoogle Scholar
  22. 22.
    DiCarlo JE, Norville JE, Mali P, Rios X, Aach J, Church GM (2013) Genome engineering in Saccharomyces cerevisiae using CRISPR-Cas systems. Nucleic Acids Res 41:4336–4343CrossRefPubMedPubMedCentralGoogle Scholar
  23. 23.
    Cong L, Zhang F (2015) Genome engineering using CRISPR-Cas9 system. Methods Mol Biol 1239:197–217CrossRefPubMedGoogle Scholar
  24. 24.
    Wu Y, Liang D, Wang Y, Bai M, Tang W, Bao S, Yan Z, Li D, Li J (2013) Correction of a genetic disease in mouse via use of CRISPR-Cas9. Cell Stem Cell 13:659–662CrossRefPubMedGoogle Scholar
  25. 25.
    Fu Y, Foden JA, Khayter C, Maeder ML, Reyon D, Joung JK, Sander JD (2013) High-frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells. Nat Biotechnol 31:822–826CrossRefPubMedPubMedCentralGoogle Scholar
  26. 26.
    Pattanayak V, Lin S, Guilinger JP, Ma E, Doudna JA, Liu DR (2013) High-throughput profiling of off-target DNA cleavage reveals RNA programmed Cas9 nuclease specificity. Nat Biotechnol 31:839–843CrossRefPubMedPubMedCentralGoogle Scholar
  27. 27.
    Xiao A, Cheng Z, Kong L, Zhu Z, Lin S, Gao G, Zhang B (2014) CasOT: a genome-wide Cas9/gRNA offtarget searching tool. Bioinformatics 30:1180–1182CrossRefGoogle Scholar
  28. 28.
    Slaymaker IM, Gao L, Zetsche B, Scott DA, Yan WX, Zhang F (2016) Rationally engineered Cas9 nucleases with improved specificity. Science 351:84–88CrossRefPubMedGoogle Scholar
  29. 29.
    Shahar OD, Raghu Ram EV, Shimshoni E, Hareli S, Meshorer E, Goldberg M (2012) Live imaging of induced and controlled DNA double-strand break formation reveals extremely low repair by homologous recombination in human cells. Oncogene 31:3495–3504CrossRefPubMedGoogle Scholar
  30. 30.
    Chang N, Sun C, Gao L, Zhu D, Xu X, Zhu X, Xiong JW, Xi JJ (2013) Genome editing with RNA-guided Cas9 nuclease in zebrafish embryos. Cell Res 23:465–472CrossRefPubMedPubMedCentralGoogle Scholar
  31. 31.
    Miller JC, Tan S, Qiao G, Barlow KA, Wang J, Xia DF, Meng X, Paschon DE, Leung E, Hinkley SJ, Dulay GP, Hua KL, Ankoudinova I, Cost GJ, Urnov FD, Zhang HS, Holmes MC, Zhang L, Gregory PD, Rebar EJ (2011) A TALE nuclease architecture for efficient genome editing. Nat Biotechnol 29:143–148CrossRefPubMedGoogle Scholar
  32. 32.
    Miller JC, Holmes MC, Wang J, Guschin DY, Lee YL, Rupniewski I, Beausejour CM, Waite AJ, Wang NS, Kim KA, Gregory PD, Pabo CO, Rebar EJ (2007) An improved zinc-finger nuclease architecture for highly specific genome editing. Nat Biotechnol 25:778–785CrossRefPubMedGoogle Scholar
  33. 33.
    Kim H, Kim MS, Wee G, Lee CI, Kim H, Kim JS (2013) Magnetic separation and antibiotics selection enable enrichment of cells with ZFN/TALEN-induced mutations. PLoS One 8:e56476CrossRefPubMedPubMedCentralGoogle Scholar
  34. 34.
    Grobarczyk B, Franco B, Hanon K, Malgrange B (2015) Generation of Isogenic Human iPS Cell Line Precisely Corrected by Genome Editing Using the CRISPR/Cas9 System. Stem Cell Rev Rep 11:774–787CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  • Jing Zhou
    • 1
  • Chencheng Wang
    • 1
  • Kunshan Zhang
    • 2
  • Yingying Wang
    • 2
  • Xi Gong
    • 1
  • Yanlu Wang
    • 1
  • Siguang Li
    • 2
    Email author
  • Yuping Luo
    • 1
    • 2
    Email author
  1. 1.College of Life SciencesNanchang UniversityNanchangChina
  2. 2.Stem Cell Translational Research Center, Tongji HospitalTongji University School of MedicineShanghaiChina

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