Tetracycline‐Inducible and Reversible Stable Gene Expression in Human iPSC‐Derived Neural Progenitors and in the Postnatal Mouse Brain

Aslam Abbasi Akhtar1, Joshua J. Breunig2

1 Department of Biomedical Sciences, Cedars‐Sinai Medical Center, Los Angeles, California, 2 Department of Medicine, UCLA, Los Angeles, California
Publication Name:  Current Protocols in Stem Cell Biology
Unit Number:  Unit 5A.9
DOI:  10.1002/cpsc.28
Online Posting Date:  May, 2017
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Abstract

The pB‐tet‐GOI plasmid system allows for stable piggyBac transposition‐mediated integration into cells, a fluorescent nuclear reporter to identify cells that have been transfected, and robust transgene activation or suppression upon the addition of dox to the cell culture or diet of the animal. Furthermore, the addition of luciferase downstream of the target gene allows for quantitative assessment of gene activity in a non‐invasive manner. The protocols herein provide instructions for the use of this system in cell lines and in the neonatal mouse brain. Specifically, a detailed protocol is provided to illustrate: (1) cloning of the respective GOI (genetic element(s) of interest); (2) nucleofection of the plasmid system into human induced pluripotent stem cell (iPSC)‐derived neural progenitors; (3) dox‐induced activation in vitro or in vivo; and (4) non‐invasive assessment of gene activity in vivo by bioluminescence imaging. © 2017 by John Wiley & Sons, Inc.

Keywords: piggyBac; transactivator; dox; tet; tetracycline; inducible; reversible

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Cloning of Respective GOI (Genetic Element of Interest) Into Response Plasmid
  • Basic Protocol 2: In Vitro Nucleofection of iPSC‐Derived Human/Mouse Neural Progenitor Cells and Subsequent Derivation of Stable Inducible Cell Lines
  • Basic Protocol 3: Adding Doxycycline to Cells to Induce/Reverse GOI
  • Basic Protocol 4: Assessing Gene Expression In Vivo by Non‐Invasive Bioluminescence Imaging of Luciferase Activity
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Cloning of Respective GOI (Genetic Element of Interest) Into Response Plasmid

  Materials
  • Three plasmids of pB‐tet‐GOI system:
    • pCAG‐pBase: pBase plasmid that constitutively expresses pBase protein
    • pCAG‐rtTA‐v10‐pB: A transactivator plasmid that constitutively expresses the rtTA‐V10 transactivator
    • pCAG‐TagBFPv5nls‐TRE‐Bi‐Clover‐Luc/GOI‐pB: Response plasmid, which has constitutive expression of TagBFPv5nls (Blue fluorescence protein with a V5 tag and nuclear localization sequence) and inducible expression of membrane clover (GFP variant) and luciferase along with a GOI (genetic element of interest) from the bi‐directional (Bi) tet response element (TRE)
  • Restriction enzymes
  • Infusion, assembly (e.g. Gibson or NEB), or ligation enzyme + respective buffer or kit in the case of Gibson or NEB assembly
  • Maxi Prep Kit (Macherey‐Nagel, cat. no. 740420.50)

Basic Protocol 2: In Vitro Nucleofection of iPSC‐Derived Human/Mouse Neural Progenitor Cells and Subsequent Derivation of Stable Inducible Cell Lines

  Materials
  • Νucleofection kit (Lonza, cat. no. VPG‐1004) containing:
    • Nucleofection reaction
    • pBase plasmid, transactivator plasmid, and response plasmid (from protocol 1, Fig. )
  • Human iPSC‐derived NPCs (Ebert et al., ; or desired cell type)
  • TrypLE Express (Gibco, cat. no. 12604013) or desired dissociation reagent
  • Phosphate‐buffered saline (PBS; Sigma, cat. no. D8662)
  • Medium for human iPSC‐derived neural progenitor cells (see recipe)
  • Cell scraper, optional
  • 37°C incubator
  • 15‐ml conical tubes
  • Centrifuge
  • Hemacytometer
  • Disposable cotton‐plugged borosilicate‐glass Pasteur pipettes (Fisher 13‐678‐8B)
  • 1‐ml pipettes
  • 70‐µm cell strainer (Falcon, cat. no. 08‐771‐2)
  • Lonza Nucleofector 2b device and corresponding solutions/cuvettes (specific to cell type being nucleofected)
NOTE: The data presented in this report did not involve staining as native fluorescence was imaged. However, the following antibodies have been verified for staining V5, clover, and luciferase using our system in mouse and human neural progenitor cells.
  • Antibodies include: Chicken anti‐EGFP 1:5000 (Abcam, cat. no. 13970),
  • Goat anti‐V5 1:1000 (Abcam, cat. no. 95038),
  • Mouse anti‐V5 1:1000 (Invitrogen, cat. no. 46‐0705), and
  • Rabbit anti‐luciferase 1:1000 (Abcam, cat. no. 21176).

Basic Protocol 3: Adding Doxycycline to Cells to Induce/Reverse GOI

  Materials
  • Doxycycline (dox; Clontech, cat. no. 631311)
  • Cell culture growth medium (from protocol 2; see recipe)
  • Phosphate‐buffered saline (PBS; Sigma, cat. no. D8662)
  • Mice (CD1 mice used for experiments; we do not foresee issues with the use of other stains)
  • Dark tubes or clear tubes wrapped in aluminum foil
  • Re‐useable feeding needle (FST, cat. no. 18060‐20)
  • Disposable feeding needle (Instech, cat. no. FTP‐20‐30)
  • 1‐ml syringe (BD, cat. no. 309659)
  • Animal scale
  • Animal cages

Basic Protocol 4: Assessing Gene Expression In Vivo by Non‐Invasive Bioluminescence Imaging of Luciferase Activity

  Materials
  • VivoGlo Luciferin (Promega, cat. no. 1043)
  • 10 mM HEPES, pH7.5 (Sigma, cat. no. H0887‐100ML, dilute and pH)
  • Mice to be imaged (see protocol 3)
  • Isoflurane chamber
  • 1‐ml tubes
  • Animal scale
  • Animal shaver
  • 1/2‐ml syringe with attached 27‐G needle (BD, cat. no. 305620)
  • Animal cages
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Figures

Videos

Literature Cited

Literature Cited
  Ables, J. L., Breunig, J. J., Eisch, A. J., & Rakic, P. (2011). Not(ch) just development: Notch signalling in the adult brain. Nature Reviews Neuroscience, 12, 269–283. doi: 10.1038/nrn3024.
  Akhtar, A. A., & Breunig, J. J. (2015). Lost highway(s): Barriers to postnatal cortical neurogenesis and implications for brain repair. Frontiers in Cellular Neuroscience, 9, 216. doi: 10.3389/fncel.2015.00216.
  Akhtar, A. A., Molina, J., Dutra‐Clarke, M., Kim, G. B., Levy, R., Schreiber‐Stainthorp, W., … Breunig, J. J. (2015). A transposon‐mediated system for flexible control of transgene expression in stem and progenitor‐derived lineages. Stem Cell Reports, 4, 323–331. doi: 10.1016/j.stemcr.2015.01.013.
  Behrstock, S., Ebert, A., McHugh, J., Vosberg, S., Moore, J., Schneider, B., … Svendsen, C. N. (2006). Human neural progenitors deliver glial cell line‐derived neurotrophic factor to parkinsonian rodents and aged primates. Gene Therapy, 13, 379–388. doi: 10.1038/sj.gt.3302679.
  Braun, S. M., Machado, R. A., & Jessberger, S. (2013). Temporal control of retroviral transgene expression in newborn cells in the adult brain. Stem Cell Reports, 1, 114–122. doi: 10.1016/j.stemcr.2013.06.003.
  Breunig, J. J., Levy, R., Antonuk, C. D., Molina, J., Dutra‐Clarke, M., Park, H., … Danielpour, M. (2015). Ets factors regulate neural stem cell depletion and gliogenesis in ras pathway glioma. Cell Reports, 12(2), 258–271. doi: 10.1016/j.celrep.2015.06.012.
  Carleton, A., Petreanu, L. T., Lansford, R., Alvarez‐Buylla, A., & Lledo, P. M. (2003). Becoming a new neuron in the adult olfactory bulb. Nature Neuroscience, 6, 507–518. doi: 10.1038/nn1048.
  Chen, F., & LoTurco, J. (2012). A method for stable transgenesis of radial glia lineage in rat neocortex by piggyBac mediated transposition. Journal of Neuroscience Methods, 207, 172–180. doi: 10.1016/j.jneumeth.2012.03.016.
  Chtarto, A., Humbert‐Claude, M., Bockstael, O., Das, A. T., Boutry, S., Breger, L. S., … Tenenbaum, L. (2016). A regulatable AAV vector mediating GDNF biological effects at clinically‐approved sub‐antimicrobial doxycycline doses. Molecular Therapy Methods & Clinical Development, 5, 16027. doi: 10.1038/mtm.2016.27.
  Ebert, A. D., Shelley, B. C., Hurley, A. M., Onorati, M., Castiglioni, V., Patitucci, T. N., … Svendsen, C. N. (2013). EZ spheres: A stable and expandable culture system for the generation of pre‐rosette multipotent stem cells from human ESCs and iPSCs. Stem Cell Research, 10, 417–427. doi: 10.1016/j.scr.2013.01.009.
  Levy, R., Molina, J., Danielpour, M., & Breunig, J. J. (2014). Neonatal pial surface electroporation. Journal of Visualized Experiments. doi: 10.3791/51319.
  Loulier, K., Barry, R., Mahou, P., Le Franc, Y., Supatto, W., Matho, K. S., … Livet, J. (2014). Multiplex cell and lineage tracking with combinatorial labels. Neuron, 81, 505–520. doi: 10.1016/j.neuron.2013.12.016.
  Mattis, V. B., Tom, C., Akimov, S., Saeedian, J., Ostergaard, M. E., Southwell, A. L., … Svendsen, C. N. (2015). HD iPSC‐derived neural progenitors accumulate in culture and are susceptible to BDNF withdrawal due to glutamate toxicity. Human Molecular Genetics, 24, 3257–3271. doi: 10.1093/hmg/ddv080.
  Morimoto, M., & Kopan, R. (2009). rtTA toxicity limits the usefulness of the SP‐C‐rtTA transgenic mouse. Developmental Biology, 325, 171–178. doi: 10.1016/j.ydbio.2008.10.013.
  Moullan, N., Mouchiroud, L., Wang, X., Ryu, D., Williams, E. G., Mottis, A., … Auwerx, J. (2015). Tetracyclines disturb mitochondrial function across eukaryotic models: A call for caution in biomedical research. Cell Reports. doi: 10.1016/j.celrep.2015.02.034.
  Suzuki, M., McHugh, J., Tork, C., Shelley, B., Klein, S. M., Aebischer, P., & Svendsen, C. N. (2007). GDNF secreting human neural progenitor cells protect dying motor neurons, but not their projection to muscle, in a rat model of familial ALS. PloS One, 2, e689. doi: 10.1371/journal.pone.0000689.
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