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Nov 2019

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Rapid Generation of Human Neuronal Cell Models Enabling Inducible Expression of Proteins-of-interest for Functional Studies
快速建立能诱导表达功能研究所需蛋白的人神经细胞模型   

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Abstract

CRISPR-Cas9 technology has transformed the ability to edit genomic sequences and control gene expression with unprecedented ease and scale. However, precise genomic insertions of coding sequences using this technology remain time-consuming and inefficient because they require introducing adjacent single-strand cuts through Cas9 nickase action and invoking the host-encoded homology-directed repair program through the concomitant introduction of large repair templates. Here, we present a system for the rapid study of any protein-of-interest in two neuronal cell models following its inducible expression from the human AAVS1 safe harbor locus. With lox-flanked foundation cassettes in the AAVS1 site and a tailor-made plasmid for accepting coding sequences-of-interest in place, the system allows investigators to produce their own neuronal cell models for the inducible expression of any coding sequence in less than a month. Due to the availability of preinserted enhanced green fluorescent protein (EGFP) coding sequences that can be fused to the protein-of-interest, the system facilitates functional investigations that track a protein-of-interest by live-cell microscopy as well as interactome analyses that capitalize on the availability of exquisitely efficient EGFP capture matrices.

Keywords: Human neuronal cells (人神经元细胞), CRISPR-Cas9 (CRISPR-Cas9), AAVS1 (AAVS1), Inducible expression (诱导表达), Protein-of-interest (目标蛋白), EGFP (增强绿色荧光蛋白)

Background

The ability to engineer the genomes of cell models and organisms holds tremendous potential for research and targeted therapy. With the advent of CRISPR-Cas9 technology, the efficiency of precision genome engineering has greatly improved, and useful reagents are coming online at a rapid pace. Despite these advances, there still is a scarcity of cell-based in vitro paradigms for studying neurological diseases that is holding back the pace of progress in this area. Whereas CRISPR-Cas9 mediated homology-directed repair (HDR) can now easily be applied to generate knockout models or to knock in small transgenic segments, the goal to integrate larger segments continues to present a considerable challenge (Devkota, 2018).

In our own work, we often would like to learn more about the function of a protein by studying its subcellular localization and protein-protein interactions. To this end, it would help if neuronal cell models could be equipped with inducible expression systems that provide temporal control of the expression of a protein-of-interest and facilitate its visualization and capture. Not long ago, this need would call for the stable transfection or lentiviral transduction of an expression cassette, an approach that can result in unpredictable confounding effects due to the random integration of transgenes. CRISPR-Cas9 provides alternative strategies to accomplish this task in a precise way but these approaches remain time-consuming and require considerable investments in resources and expertise.

To address this unmet need, we sought to develop resources that enable the rapid and flexible integration of large inducible expression cassettes into the AAVS1 human safe harbor locus (DeKelver et al., 2010) of several neuronal cell models. Stable insertion into the AAVS1 locus, as opposed to viral integration, was chosen to preclude transgene silencing and insertional mutagenesis, notorious confounders associated with non-directed transgene insertions. To maximize flexibility and speed, the system was built around two genome-editing steps. The first step, which makes use of CRISPR-Cas9-mediated HDR followed by selection of positive clones, is time-consuming and uncertain to succeed but we have already accomplished this in 3 cell lines that are available to be shared. The second step, which relies on a Cre recombinase-mediated exchange of an expression cassette, is relatively easy to accomplish and fast because it can be followed by puromycin-based selection of positive clones.

More specifically, a paired Cas9 nickase CRISPR strategy, used to improve the specificity of targeting and to minimize off-target effects (Ran et al., 2013), was employed to insert a foundation cassette (FC), comprising a pair of lox sites flanking a G418 resistance marker into Intron 1 of the AAVS1 locus, a human genetic safe harbour. In parallel, a ~7 kb inducible expression plasmid (IEX) was built. The expression cassette within the IEX is flanked by compatible lox sites and comprises elements designed to accept the insertion and control the expression of any coding sequence-of-interest (Figure 1). More specifically, the IEX comprises a promoter-less puromycin resistance marker, the reverse transactivator (rtTA3), and an EGFP tag fused to a protein-of-interest insertion site. With this arrangement, the rtTA3 is designed to drive the inducible expression of the EGFP-fused protein-of-interest that is under the control of the TREtight inducible promoter.

With these tools in hand, novel cell models for the inducible expression of a protein-of-interest fused to EGFP can be generated in three straightforward steps:
1. Insert coding sequence for protein-of-interest into the IEX plasmid.
2. Transiently co-transfect the IEX plasmid and a plasmid coding for Cre recombinase into the parental cell equipped with one or two FCs.
3. Select stable integrant clones by puromycin selection or fluorescence activated cell sorting (FACS).

In addition to its ease of handling and speed, the system restricts the copy number of insertions to two, corresponding to the two AAVS1 alleles present in the human genome. The system also lends itself to comparative analyses of several proteins, because its design ensures that cells coding for different proteins-of-interest can be generated side-by-side, requiring no specific resources, except for a human cDNA library and the corresponding primers to amplify and insert a given coding sequence into the IEX cassette. Note that fusing the coding sequence for a protein-of-interest to EGFP does not only provide the ability to undertake live cell imaging but the EGFP tag also allows the high affinity purification of fusion proteins by GFP nanotrap technology for the study of protein-protein interactions (Rothbauer et al., 2008).

We recently applied the system to study the TAU protein and BAG5 in several human neuronal cell models, including ReN VM human neural progenitor cells that can be differentiated into a co-culture of neural and glial cells (Donato et al., 2007). These studies have led us to detect subtle differences in protein-protein interactions of wild-type versus mutant forms of TAU and BAG5 (Figure 1). This article was written with the intent to share these resources and facilitate their adaptation to the study of other proteins-of-interest.



Figure 1. Design of AAVS1 embedded system for inducible expression of proteins-of-interest. A. CRISPR-Cas9 nickase-mediated insertion of foundation cassette (FC) into AAVS1 safe harbour. B. Insertion of coding sequence-of-interest into inducible expression plasmid (IEX). C. Reverse-tetracycline-controlled transactivator (rtTA3)-mediated expression of protein-of-interest following addition of doxycycline to cell culture medium. D. Example 1: Parallel induction of wild-type and P301L mutant TAU fused to C-terminal EGFP in ReN cells. Antibodies directed against beta-actin served as the loading control. E. Example 2: Side-by-side induction of N-terminally EGFP-tagged wild-type or mutant (DARA) BAG5. Cells expressing EGFP only served as additional controls in this analysis.

Materials and Reagents

  1. Biological materials
    1. Plasmids (Table 1)
    2. Cell lines (Table 2)

      Table 1. List of available plasmids for generating inducible cells and their Addgene ID


      Table 2. List of available cell lines


  2. Cloning
    Note: All cloning enzymes and PCR reagents are stored at -20 °C, stable for 1 year.
    1. Q5® Hot Start high-fidelity 2x master mix (New England Biolabs, catalog number: M0494S )
    2. Primers: ordered as Value Oligos or Custom Oligos, 25 nmole, desalted (Invitrogen). Reconstitute to 100 μM stocks in water, store at -20 °C
    3. Q5® Site-directed mutagenesis kit (New England Biolabs, catalog number: E0554S )
    4. EZ-10 spin column PCR products purification kit (BioBasic, catalog number: BS664-250preps )
    5. Restriction enzyme BbvCI with CutSmart buffer (New England Biolabs, catalog number: R0601S )
    6. Restriction enzyme NheI-HF with CutSmart buffer (New England Biolabs, CutSmart, catalog number: R3131S )
    7. Fast AP thermosensitive alkaline phosphatase (Thermo Fisher Scientific, catalog number: EF0651 )
    8. PureLink Quick Gel Extraction Kit (Invitrogen, catalog number: K210012 )
    9. T4 DNA ligase (New England Biolabs, catalog number: M0202S )
    10. EnduraTM Chemically Competent E. coli Cells (Lucigen, catalog number: 60241-2 ). Store at
      -80 °C
    11. EZ-10 spin column plasmid DNA minipreps kit (BioBasic, catalog number: BS614-250preps )
    12. PureLink® HiPure plasmid filter maxi kit (Invitrogen, catalog number: K210026 )
    13. Ampicillin sodium salt powder, 91.0-100.5%, cell culture tested (Sigma-Aldrich, catalog number: A1066-5G ). Store at 4 °C, stable for 1 year

  3. Inducible cell generation
    1. paavCAG-iCre plasmid (Addgene, catalog number: 51904 , a gift from Jinhyn Kim)
    2. jetPRIME (PolyPlus, catalog number: 114-07 ). Store at 4 °C, stable for 1 year
    3. TransfeX transfection reagent (ATCC, catalog number: ACS-4005 ). Store at 4 °C, stable for 1 year
    4. Millex-HA syringe filters, 0.45 μm pore size (Millipore, catalog number: 5010-SLHA033SS )
    5. Millex-GP syringe filters, 0.22 μm pore size (Millipore, catalog number: SLGP033RS )
    6. 6-well tissue-culture plates (Falcon, catalog number: 353046 )
    7. 24-well tissue-culture plates (Falcon, catalog number: 353047 )
    8. Puromycin (Sigma-Aldrich, catalog number: P7255-25MG ). Reconstitute to 10 mg/ml stock, filter sterilize, aliquot, store at -20 °C
    9. Doxycycline (BioBasic, catalog number: DB0889-25G ). Reconstitute to 10 mg/ml stock, aliquot, store at -20 °C

  4. Affinity capture with GFP nanotrap
    1. 15-cm tissue-culture plates (Sarstedt, catalog number: 83.3903 )
    2. Agarose-conjugated GFP nanotrap (Chromotek, catalog number: gta-10 ). Store at 4 °C, stable for 1 year

  5. Cell culture
    1. 6-cm tissue-culture plates (Sarstedt, catalog number: 83.3901 )
    2. 1.5 ml centrifuge tubes (Sarstedt, catalog number: 72.690.300 )
    3. 12-well tissue-culture plates (Sarstedt, catalog number: 83.3921 )
    4. Dulbecco's Phosphate-Buffered Saline (D-PBS), without calcium chloride and magnesium chloride, liquidPhosphate buffered saline (Sigma-Aldrich, catalog number: D8537-500ML ). Store at 4 °C, stable for 1 year5.
    5. 0.25% Trypsin with EDTA 4Na (Gibco, catalog number: 25200072 ). Aliquot, store at -20 °C for long-term storage. Store at 4 °C for short-term use, stable for 6 months
    6. StemPro® Accutase® Cell Dissociation Reagent (Gibco, catalog number: A1110501 ). Aliquot, store at -20 °C for long-term storage. Store at 4 °C for short-term use, stable for 6 months
    7. Matrigel, growth-factor reduced (Corning, catalog number: 354230 ). Aliquot, store at -20 °C, stable for 1 year
    8. RecoveryTM Cell Culture Freezing Medium (Gibco, catalog number: 12648010 ). Aliquot, store at -20 °C, stable for 1 year
    9. DMEM with high glucose, L-glutamine, and sodium pyruvate (Gibco, catalog number: 11995073 ). Store at 4 °C, stable for 1 year
    10. 10% Fetal Bovine Serum, qualified (Gibco, catalog number: 12483020 ). Aliquot, store at -20 °C for long-term storage
    11. 1% GlutaMAXTM-I supplement (Gibco, catalog number: 35050061 ). Store at 4 °C, for stable for 1 year
    12. DMEM/F12, 1:1, contains L-glutamine, but no HEPES (Gibco, catalog number: 21041025 ). Store at 4 °C, stable for 1 year
    13. 2% N21-MAX supplement (R&D Systems, catalog number AR008 ) or 2% B-27 serum-free supplement (Gibco, catalog number: 17504044 ). Store at -20 °C, stable for 1 year
    14. Basic fibroblast growth factor (Gibco, catalog number: PHG0261 ). Store at -80 °C, stable for 1 year
    15. Epidermal growth factor (Reprokine, catalog number: RKP01133 ). Store at -80 °C, stable for 1 year
    16. Heparin (Sigma-Aldrich, catalog number: H3149-10KU ). Store at -80 °C, stable for 1 year
    17. 2 ng/ml glial-derived neurotrophic factor (Gibco, catalog number: PHC7045 )
    18. 500 μM dibutyryl-cyclic-adenosine monophosphate (Sigma-Aldrich, catalog number: D0627-250MG )
    19. Proliferation media for IMR-32 and HEK-293 cells (see Recipes)
    20. Proliferation media for ReN VM cells (see Recipes)
    21. Neuronal differentiation media for ReN VM cells (see Recipes)

  6. Cell lysis
    Note: Original reagents are stored at room temperature. Reconstituted stocks are stored at 4 °C for 3 months. All buffers (see Recipes) are prepared fresh from reagent stocks immediately before use.
    1. Tris-HCl, > 99% (BioShop, catalog number: TRS002.5 ). Reconstitute to 1 M stock in water
    2. NP-40 (BioShop, catalog number: NON505.500 ). Reconstitute to 10% stock in water
    3. Sodium deoxycholate (DOC), > 99% (BioShop, catalog number: DCA333.50 ). Reconstitute to 10% w/v stock in water, pH 8.0
    4. Sodium chloride (NaCl), > 99% (BioShop, catalog number: SOD002.205 ). Reconstitute to 5 M stock in water
    5. Ethylenediaminetetraacetate (EDTA), >99% (BioShop, catalog number: EDT001.500 ). Reconstitute to 0.5 M stock in water
    6. Sodium orthovanadate (Na3VO4) (BioShop, catalog number: SOV664.25 ). Reconstitute to 0.1 M stock in water
    7. Sodium fluoride (NaF) (BioShop, catalog number: SFL001.100 ). Reconstitute to 1 M stock in water
    8. Phenylmethylsulfonyl fluoride (PMSF) (BioShop, catalog number: PMS123.5 ). Reconstitute to 0.5 M stock in ethanol
    9. cOmplete protease inhibitor (Roche, catalog number: 11836170001 )
    10. PhosStop phosphatase inhibitor (Roche, catalog number: 4906837001 )
    11. HEPES (Bioshop, catalog number: HEP001.500 ). Reconstitute to 0.1 M stock in water
    12. Trifluoroacetic acid (TFA), > 99%, HPLC grade (Sigma-Aldrich, catalog number: 302031-100ML )
    13. Acetonitrile (ACN), HPLC grade (Caledon, catalog number: 1401-7-40 )
    14. Lysis buffer (see Recipes)
    15. Wash buffer (see Recipes)
    16. Elution buffer (see Recipes)

Equipment

  1. -80 °C freezer (Thermo Fisher, model: Standard Performance Ultra-Low Freezers )
  2. Refrigerated micro centrifuge (Eppendorf, model: 5424R , catalog number: 5404000138 )
  3. Avanti J-26S series high-speed centrifuge (Beckman-Coulter, catalog number: B22984 )
  4. Orbital shaker (Jeio Tech, model: ISF-7000 series )
  5. Fluorescence microscope (Leica Microsystems, model: Leica DMI 6000 B )
  6. NanoDrop spectrophotometer (Thermo Fischer Scientific, catalog number: ND-1000 )
  7. ESBE Scientific Save cell UV cell culture incubator (Panasonic, catalog number: MCO-19AIC )
  8. SterilGARD biosafety cabinet (The Baker Company, catalog number: SG603A-HE )

Software

  1. Snapgene (GSL Biotech LLC, https://www.snapgene.com/)
  2. Snapgene Viewer (GSL Biotech LLC, https://www.snapgene.com/snapgene-viewer/)
  3. Tm Calculator (New England Biolabs, https://tmcalculator.neb.com/#!/main)
  4. NEBioCalculator (New England Biolabs, https://nebiocalculator.neb.com/#!/ligation)
  5. NanoDrop 1000 (Thermo Fischer Scientific, https://www.thermofisher.com/ca/en/home/industrial/spectroscopy-elemental-isotope-analysis/molecular-spectroscopy/ultraviolet-visible-visible-spectrophotometry-uv-vis-vis/uv-vis-vis-instruments/nanodrop-microvolume-spectrophotometers/nanodrop-software-download.html)

Procedure

The protocol is designed to enable the generation of neural cell models for functional analyses of proteins-of-interest in less than a month (Figure 2). The system is scalable by parallelization of procedures.


Figure 2. Time-line for the execution of procedures described in this protocol. All steps described in the protocol take approximately one month to implement. The generation of vectors coding for the inducible expression of a protein-of-interest can be completed in approximately one week. Once positive clones have been selected and validated, cell-based and biochemical analyses can be undertaken in parallel. Naturally, the one-month timeline will be exceeded, if complex follow-on downstream analyses are planned.


  1. Week 1. Cloning a gene of interest into the IEX
    Notes:
    1. Cloning strategies can be simulated, verified, and visualized using Snapgene and Snapgene Viewer.
    2. The coding sequence (CDS) of a new protein-of-interest can be cloned into the IEX using the unique restriction enzyme sites BbvCI and KpnI:
      BbvCI: located 5’ of the start codon of new CDS.
      KpnI: present in the linker region connecting EGFP to the C-terminus of the new protein.
    3. Multiple other unique restriction enzyme sites (e.g., NheI, BstBI, KflI, etc.) may also be used.

    1. Order primers to append BbvCI and KpnI restriction enzyme sites to the 5’ and 3’ ends of the CDS of the protein-of-interest, respectively.
      Example:
      Primer name
      Sequence
      Insert BbvCI site 5′…CCV TCAGC…3′
      5′ N8CCTCAGCGCCACCATGX20-25 3′
      Insert KpnI site 5′…GGTAV CC…3′
      5′ N8GGTACCX20-25 3′
      1. N8 represent 8 random nucleotides to serve as overhangs required by restriction enzymes for efficient digestion.
      2. X20-25 represent sequences of the CDS of the protein-of-interest.
      3. Blue: Kozak sequence, including the ATG start codon, required for translation.
      4. Do not include a stop codon in the X20-25 sequence used for designing the ‘Insert KpnI’ primer if the new protein is to be expressed as an EGFP fusion protein.
    2. Determine the annealing temperature of primers using the New England Biolabs Tm Calculator tool, which returns this information when primer sequences are entered into the online form field at: https://tmcalculator.neb.com/#!/main.

    Day 1. Cloning
    1. Perform PCR to append BbvCI and KpnI sites to CDS of protein-of-interest using the following PCR reaction:
      Reagents
      50 μl reaction (µl)
      Final concentration
      Q5 HiFi Hot start master mix (2x)
      25
      1x
      Template DNA (20 ng)
      Calculate

      ‘Insert BbvCI’ primer (10 μM)
      2.5
      0.5 µM
      ‘Insert KpnI’ primer (10 μM)
      2.5
      0.5 µM
      Nuclease-free H2O
      Add to 50

      Total 
      50
    2. Perform the following PCR cycles:

      Step
      Temperature (°C)
      Time

      Initial denaturation
      98
      30 s

      Denaturation
      98
      10 s
      30 cycles
      Annealing
      To be determined
      30 s

      Extension
      72
      10-20 s/kb

      Final extension
      72
      2 min

      Hold
      4
      Until sample storage
    3. Proceed to PCR purification per manufacturer’s protocol. Measure DNA concentration on NanoDrop 1000 as per the manufacturer’s protocol. 
    4. Set up restriction digest of IEX vector and new CDS insert using the following reactions:
      Reagents
      IEX vector
      New CDS insert
      BbvCI
      4 µl
      4 µl
      KpnI
      4 µl
      4 µl
      CutSmart buffer (10x)
      5 µl
      5 µl
      Alkaline phosphatase
      1 µl
      --
      DNA
      Up to 5 µg
      Up to 5 µg
      Nuclease-free H2O
      Add to 50 µl
      Add to 50 µl
      Total
      50 µl
      50 µl
    5. Incubate at 37 °C for 1 h.
    6. During incubation, cast a 1% agarose gel to be used for isolating the digested products.
    7. Undertake gel electrophoresis with the entire volume of the restriction digest mixture.
      Troubleshoot: it is advised to include a sample of the uncut and singly-cut IEX vector as controls.
    8. Excise the doubly digested IEX vector (7.0 kb) and new CDS insert.
    9. Proceed to gel extraction purification per manufacturer’s protocol. Measure DNA concentration.

    Day 1. Ligation of new CDS insert into doubly digested IEX vector
    1. Determine the appropriate vector: insert mass ratios to achieve an approximate 1:1 molar ratio. This task can be simplified by using online algorithms, including the NEBioCalculator tool (version 1.10.0; https://nebiocalculator.neb.com/#!/ligation).
    2. Set up the following ligation reactions:
      Reagents
      Vector-only control (µl)
      Vector + Insert (µl)
      T4 DNA ligation buffer (10x)
      2
      2
      T4 DNA ligase
      2
      2
      Vector (100 ng)
      Calculate
      Calculate
      Insert (x ng, depends on ratio)
      --
      Calculate
      Nuclease-free H2O
      Add to 20
      Add to 20
      Total
      20
      20
    3. Incubate at room temperature for 15 min.
    4. Proceed to bacterial transformation per manufacturer’s protocol.
    5. Plate bacteria on agar plates with 100 µg/ml ampicillin (the same concentration is used for all applications involving ampicillin), incubate plates upside down at 30 °C overnight.
      Endura chemically competent cells are used to minimize truncation of complex plasmids at 30 °C growth temperature on agar. Agar plates are incubated upside down to reduce evaporation of moisture from the agar during overnight incubations.

    Day 2. Pick transformant colonies
    1. Compare the number of colonies on the ‘vector-only’ control plate, which should have none to very few colonies, and the ‘vector plus insert’ plate, which should have many colonies.
    2. Pick 6-10 colonies from the ‘vector plus insert’ plate to grow in 3 ml of LB medium with ampicillin with shaking at 225 rpm overnight at 37 °C.

    Day 3. Extract DNA for sequencing
    1. Save 500 µl of the overnight bacteria culture as a glycerol stock.
    2. Extract the DNA from the remaining bacteria culture by the miniprep method as per manufacturer’s protocol.
    3. Measure the DNA concentration and send the purified DNA plasmid for sequencing.
    4. If additional site-directed mutagenesis is required, perform the reaction using the Q5® Site-directed mutagenesis kit according to manufacturer’s protocol.

    Days 4-5. Maxiprep new IEX and iCre vectors for transfection
    1. Use the bacteria stock with the correct sequencing result to grow in a 125-250 ml culture with ampicillin, then undertake a DNA maxiprep following manufacturer instructions.
      Note: It is important to use a maxiprep kit that removes endotoxins from the bacteria to minimize undesired cellular toxicity during future transfections.
    2. At the same time, maxiprep the iCre vector for future transfections.

  2. Week 2. Generating inducible cells with new IEX vector
    Next steps are designed to create inducible cells by co-transfection of the IEX vector and the iCre vector, followed by puromycin selection and confirmation of inducibility by doxycycline (Dox) treatment.

    Ahead of Day 8
    1. Culture target cells in cell culture incubators at 37 °C, 95% moisture, and 5% CO2.
    2. Perform all protocols involving mammalian cells in certified biosafety cabinets to prevent contamination and adhere to biosafety rules and regulations.
    3. Passage at least once before transfection to check that cells are growing normally as they can be quite fragile after thawing. See ‘Auxiliary information–Cell culture’ section for instructions on regular cell culture, passaging, and saving stocks.
    4. Determine the appropriate puromycin concentration to use for selection for each cell line by treating cells at various concentrations (e.g., 0.0, 0.2, 0.5, 1.0, 2.0, 5.0, and 10 µg/ml) of puromycin and selecting the lowest concentration that kills 100% of cells in 3 days.

    Day 8. Plate cells for transfection
    1. Plate all cells in media without antibiotics or antimycotics.
    2. For ReN VM cells, plate ~135,000 cells/well on Matrigel-coated 12-well plates in proliferation media without heparin. Cells should be ~50% confluent the next day.
    3. For cells that do not need to be plated on coated cell culture dishes, plate ~350,000 cells/well on 6-well plates with the expectation that they are ~80% confluent the next day.
    4. Set 1-2 wells aside as controls for effective puromycin selection; they will not be transfected.

    Day 9. Transfection
    1. Transfect cells using a ratio of 1 iCre vector: 4 IEX vectors with desired insert.
      A ratio of 1 iCre vector: 1-3 IEX vectors have also worked.
    2. For ReN VM cells, use a total of 1.0 μg of DNA with 1 µl of TransfeX transfection reagent per well following manufacturer’s instructions.
      Use proliferation media without heparin during the transfection because heparin may compromise effectiveness.
    3. For most other cells, use a total of 3 µg of DNA with jetPrime transfection reagent per well following manufacturer’s instructions.
      For IMR-32 cells, use a ratio of 1 µg DNA:3 µl jetPrime.
      For HEK-293 cells, use a ratio of 1 µg DNA:2 µl jetPrime.
    4. To minimize toxicity change media 4 h after transfection to regular proliferation media.

  3. Week 3. Puromycin selection and Dox treatment
    1. Passage transfected cells onto a few larger plates and add the appropriate amount of puromycin and 2.0 µg/ml of Dox.
      One may check for induction levels 24 h post Dox addition on a fluorescence microscope.
    2. Continue puromycin selection until all cells in the control wells have died.
      Note: It is recommended to refresh puromycin and Dox every other day to keep the inducible transgene transcriptionally active.
    3. Allow surviving cells to proliferate to sizable colonies of > 100 cells/colony.
    4. Induce cells with 2.0 µg/ml Dox; check on the fluorescence microscope for successfully induced colonies.
      Caution: Induction in ReN VM cells may not be homogenous even within the same clone and may lose induction during the course of differentiation. This is because endogenous epigenetic silencing may cause variable and mosaic expression of inducible constructs (Chanda et al., 2017). Also, if differentiation is desired, induction of ReN VM cells and other neural progenitor cell lines may be improved by beginning induction 2-4 days during the proliferation stage before the start of differentiation.
    5. Mark and transfer selected colonies with desired level of induction to new wells. Expand them to save as stocks and to validate the inducibility and identity of the protein-of-interest by Western blot.
    6. If removal of the PuroR gene is desired, transfect cells with the ‘Flpo’ optimized Flippase vector.
    7. Select cells that have lost puromycin resistance by plating duplicates of the same cell clone, one of them puromycin treated the other left untreated.
      In ReN VM cells, retaining PuroR may help with keeping the inducible transgene region transcriptionally active.

  4. Week 4. Preparing inducible cells for mass spectrometry-based interactome studies
    Note: The following steps are designed to capture EGFP-tagged inducible protein and its interactors using GFP nanotrap for downstream analysis in mass spectrometer.
    1. Culture inducible protein-of-interest-EGFP fusion cells in biological triplicates on 15-cm plates along with cells inducibly expressing EGFP only as a control against unspecific binding to the GFP nanotrap matrix.
      Note: If induced expression is very low, scale up accordingly.
    2. Induce cells for the desired length of time.
    3. Follow manufacturer’s protocol on using GFP nanotrap for affinity capture experiments.
      All procedures should be performed at 4 °C to prevent protein degradation.
    4. Cells may be lysed in RIPA buffer as per manufacturer’s protocol or buffer composed of 0.5% NP-40 and 0.25% DOC (Recipe 4).
      Remove cellular debris by centrifugation at 3,000 x g for 5 min at 4 °C.
    5. Due to its high binding capacity, 20 μl of GFP nanotrap bead slurry/biological replicate should be sufficient for mass spectrometry-based interactome studies.
    6. EGFP fusion proteins or EGFP alone may be captured on GFP nanotrap beads during 2 h incubations at 4 °C with continuous mixing with the lysate.
      Shorter nanotrap bead incubations of 5 min followed by light centrifugation may be sufficient to visualize the capture of EGFP fusion proteins.
    7. Centrifuge at 100 x g at 4 °C to collect agarose-conjugated GFP nanotrap beads and bait proteins.
    8. Wash beads 5 times in Wash buffer. Washing procedures should be done within 2 h to minimize loss of interactors.
      1. First 3 times in Washing buffer (Recipe 5). NaCl salt concentration of 150-500 mM can be used during these washing steps, depending on desired stringency.
      2. The last two washes should be 25 mM HEPES (pH 7.0) and 10 mM HEPES (pH 7.0). Tris-HCl must not be used.
    9. Elute in 0.2% trifluoroacetic acid with 20% acetonitrile in water (pH 1.9). Immediately save and flash freeze or pH neutralize a portion of the eluates (e.g., 25% of total) for downstream validation of bait protein complexes by Western blot.
      A delay in the collection of the eluate material dedicated to Western blot analysis can lead to precipitation of eluate proteins in the pH 1.9 environment, thereby compromising the Western blot analysis.
    10. Other special considerations:
      1. One may use BCA to adjust for equal protein content before affinity capture.
      2. Remember to save a fraction of the cellular lysates as ‘input’ and ‘unbound’ for Western blot analyses.

    Auxiliary information
    Cell culture
    1. After thawing cells at 37 °C, remove DMSO content from the cell stock by diluting the cell stock in 4 ml of proliferation media and centrifuging cells at 100 x g for 3 minutes at 25 °C. Remove the supernatant to retain only the cell pellet. DMSO may be toxic to cells in regular culture.
    2. Culture cells in the appropriate proliferation media for about one week to allow recovery from thawing.
    3. For ReN VM cells, do not include antibiotics or antimycotics in the proliferation media, as they will cause cell death.
    4. For ReN VM cells, please coat the tissue-culture plates with Matrigel according to the manufacturer’s protocol at least 2 h before seeding cells onto the plates. 2 h is needed for Matrigel to solidify at 37 °C in a humidified incubator.
    5. Passage cells regularly before they reach 90% confluency to prevent the adverse effects of overgrowing with the appropriate dissociation reagent. For ReN VM cells, use Accutase. For most other cell types, use trypsin.
    6. Remember to save a portion of the cells, especially valuable clones, as stocks and while using the rest for experiments or regular culture.
    7. For preserving cell stocks, resuspend cells in Cell Freezing Media in labelled cryovials. Transfer cells to -80 °C immediately and store at -80 °C for 1 day before transferring into liquid nitrogen for long term storage.

Data analysis

Information on statistical analyses as well as were to find publicly available mass spectrometry data from the TAU and BAG5 analyses was included with the article describing the first use of this system (Wang et al., 2019), with Figure 2 of the original article providing details on the design of a quantitative interactome analysis, Figure 3 describing mass spectrometry sequence coverage of the TAU protein, Figure 6 comparing the TAU interactome in IMR32 and ReN VM cells, and Figure 9 documenting results from a post-translational modification analysis of TAU produced with this system.

Recipes

  1. Proliferation media for IMR-32 and HEK-293 cells
    Note: Make media fresh, store at 4 °C, stable for 2 months.
    DMEM media with high glucose, L-glutamine, and sodium pyruvate supplemented with 10% qualified fetal bovine serum and 1% GlutaMAXTM-I supplement
  2. Proliferation media for ReN VM cells
    Note: Make media fresh, store at 4 °C, stable for 2 weeks.
    DMEM/F12, 1:1 with L-glutamine but no HEPES supplemented with 2% N21-MAX supplement or 2% B-27 serum-free supplement
    20 ng/ml basic fibroblast growth factor
    20 ng/ml epidermal growth factor
    2 ng/ml heparin
  3. Neuronal differentiation media for ReN VM cells
    Note: Make media fresh, store at 4 °C, stable for 1 month.
    DMEM/F12 1:1 with L-glutamine but no HEPES supplemented with 2% N21-MAX supplement or 2% B-27 serum-free supplement
    2 ng/ml glial-derived neurotrophic factor
    500 μM dibutyryl-cyclic-adenosine monophosphate
  4. Lysis buffer
    Note: Make fresh before use, stable at 4 °C for 1 day.
    1. Mix 0.5% (v/v) NP-40, 0.5% (w/v) DOC, Tris/HCl, pH 8.3, 5 mM EDTA, 1 mM Na3VO4, 10 mM NaF, 1 mM PMSF, 1x cOmplete protease inhibitor, and 1x PhosStop phosphatase inhibitor
    2. Prepare NP-40 and DOC as 50x stock solutions in water and warm gently to dissolve. DOC will precipitate at pH value below 8.0. Therefore, ensure that the pH exceeds this value and is buffered before adding this detergent
    If the intention is to use lysates for immunoaffinity capture of the protein-of-interest, it is advisable to prepare a larger amount of this buffer and divert some of it for the preparation of the Wash (see below) before addition of protease and phosphatase inhibitor cocktails. Also, we recommend chilling the buffer to 4 °C before adding temperature-labile protease and phosphatase inhibitor cocktails.
  5. Wash buffer
    Note: Make fresh before use, stable at 4 °C for 2 days.
    The composition of this buffer is similar to the lysis buffer with the following considerations:
    Protease and phosphatase inhibitors can be omitted.
    If samples are used for mass spectrometry downstream, the last two washes must use only 25 mM HEPES (pH 7.0) and 10 mM HEPES (pH 7.0) in deionized water. Tris-HCl must not be used.
  6. Elution buffer
    Note: Make fresh before use, stable at room temperature for 1 day.
    1. Mix 0.2% TFA and 20% acetonitrile in deionized water
    2. The pH of this solution will be 1.9 without adjustment

Acknowledgments


XW held an Ontario Graduate Scholarship. GS received funds from the Krembil Foundation, Canadian Institutes for Health Research (grant number 137651) and the Alberta Prion Research Institute (grant number 201600028). Support by the Arnold Irwin family for the purchase of a mass spectrometer was instrumental for getting started on this project. The authors gratefully acknowledge generous philanthropic support by the Borden Rosiak family.

Competing interests

The authors declare that they have no financial or non-financial competing interests.

Ethics

The study did not involve human subjects or animal work. All handling of human cell lines and chemicals were undertaken in accordance with an active biosafety protocol (number 208-S06-2) approved by the environmental health and safety (EHS) office at the University of Toronto, Toronto, Canada.

References

  1. Chanda, D., Hensel, J. A., Higgs, J. T., Grover, R., Kaza, N. and Ponnazhagan, S. (2017). Effects of cellular methylation on transgene expression and site-specific integration of adeno-associated virus. Genes (Basel) 8(9): 232.
  2. DeKelver, R. C., Choi, V. M., Moehle, E. A., Paschon, D. E., Hockemeyer, D., Meijsing, S. H., Sancak, Y., Cui, X., Steine, E. J., Miller, J. C., Tam, P., Bartsevich, V. V., Meng, X., Rupniewski, I., Gopalan, S. M., Sun, H. C., Pitz, K. J., Rock, J. M., Zhang, L., Davis, G. D., Rebar, E. J., Cheeseman, I. M., Yamamoto, K. R., Sabatini, D. M., Jaenisch, R., Gregory, P. D. and Urnov, F. D. (2010). Functional genomics, proteomics, and regulatory DNA analysis in isogenic settings using zinc finger nuclease-driven transgenesis into a safe harbor locus in the human genome. Genome Res 20(8): 1133-1142.
  3. Devkota, S. (2018). The road less traveled: strategies to enhance the frequency of homology-directed repair (HDR) for increased efficiency of CRISPR/Cas-mediated transgenesis. BMB Rep 51(9): 437-443.
  4. Donato, R., Miljan, E. A., Hines, S. J., Aouabdi, S., Pollock, K., Patel, S., Edwards, F. A. and Sinden, J. D. (2007). Differential development of neuronal physiological responsiveness in two human neural stem cell lines. BMC Neurosci 8: 36.
  5. Ran, F. A., Hsu, P. D., Lin, C. Y., Gootenberg, J. S., Konermann, S., Trevino, A. E., Scott, D. A., Inoue, A., Matoba, S., Zhang, Y. and Zhang, F. (2013). Double nicking by RNA-guided CRISPR Cas9 for enhanced genome editing specificity. Cell 154(6): 1380-1389.
  6. Rothbauer, U., Zolghadr, K., Muyldermans, S., Schepers, A., Cardoso, M. C. and Leonhardt, H. (2008). A versatile nanotrap for biochemical and functional studies with fluorescent fusion proteins. Mol Cell Proteomics 7(2): 282-289.
  7. Wang, X., Williams, D., Müller, I., Lemieux, M., Dukart, R., Maia, I. B. L., Wang, H., Woerman, A. L. and Schmitt-Ulms, G. (2019). Tau interactome analyses in CRISPR-Cas9 engineered neuronal cells reveal ATPase-dependent binding of wild-type but not P301L Tau to non-muscle myosins. Sci Rep 9(1): 16238.

简介

[摘要] CRISPR-Cas9技术以前所未有的简便性和规模改变了编辑基因组序列和控制基因表达的能力。但是,由于需要引入相邻的单链,因此使用该技术进行精确的基因组编码插入仍然很耗时且效率低下。通过减少Cas9切口酶的作用并通过同时引入大型修复模板来调用宿主编码的同源性指导的修复程序。在此,我们提出了一种系统,用于在其诱导后的两个神经元细胞模型中快速研究任何目的蛋白该系统可从人类AAVS1 安全港基因座表达,在AAVS1 位点具有lox侧翼的基础盒和定制的质粒以接受感兴趣的编码序列,该系统使研究人员能够为诱导型产生自己的神经元细胞模型任何编码表达序列不到一个月的时间。由于可用性Preinserted 增强型绿色的Fluo 可以与目标蛋白质融合的最新蛋白质(EGFP)编码序列,该系统可帮助功能研究通过活细胞显微镜以及利用非常有效的可用性进行的相互作用组分析来跟踪目标蛋白质EGFP捕获矩阵。

[背景] 工程化细胞模型和生物体基因组的能力在研究和靶向治疗方面具有巨大潜力。随着CRISPR-Cas9技术的出现,精确基因组工程的效率大大提高,有用的试剂正在网上尽管取得了这些进展,但仍然缺乏基于细胞的体外研究神经逻辑疾病的范例,这阻碍了该领域的发展步伐,而CRISPR-Cas9介导的同源指导修复(HDR)现在可以容易地用于产生基因敲除模型或敲入小的转基因片段,整合较大片段的目标仍然提出了相当大的挑战(Devkota,2018)。

在我们自己的工作中,我们通常希望通过研究蛋白质的亚细胞定位和蛋白质-蛋白质相互作用来了解蛋白质的功能。为此,如果神经元细胞模型可以配备可提供瞬时表达的诱导系统,这将有所帮助控制目的蛋白的表达并促进其可视化和捕获。不久前,这种需求将要求表达盒的稳定转染或慢病毒转导,这种方法可能会导致不可预测的混杂效应。 CRISPR-Cas9提供了替代策略以精确的方式完成此任务,但是这些方法仍然很耗时,并且需要大量的资源和专业知识投资。

为了解决这一未被满足的需求,我们寻求发展资源,可以快速,灵活集成大量诱导表达盒引入的AAVS1 人类安全港基因座(DeKelver 等,2010)几种神经细胞模型。稳定插入AAVS1 轨迹与病毒整合相反,选择该基因是为了避免转基因沉默和插入诱变,即与非定向转基因插入相关的臭名昭著的混杂因素。为了最大程度地提高灵活性和速度,该系统围绕两个基因组编辑步骤构建。使用CRISPR-Cas9介导的HDR继之以选择阳性克隆非常耗时且不确定,但我们已经在3种可共享的细胞系中实现了这一目标。第二步,依赖于Cre 重组酶介导的表达盒的交换相对容易实现和快速,因为它可以随后进行基于嘌呤霉素的阳性克隆选择。

更具体地说,采用成对的Cas9切口酶CRISPR策略来提高靶向特异性并最小化脱靶效应(Ran 等人,2013),用于插入包含一对lox位点的基础盒(FC)。在人类遗传安全港AAVS1 基因座的内含子1中侧接一个G418抗性标记,同时构建了一个约7 kb的诱导表达质粒(IEX).IEX中的表达盒两侧是相容的lox位点和突出的元件设计用于接受插入并控制任何目的编码序列的表达(图1 )。更具体地说,IEX促进了无启动子的嘌呤霉素抗性标记,反向反式激活子(rtTA 3 )和与之融合的EGFP标签蛋白的-利益插入位点。有了这种安排,RtTA3设计用于驱动诱导表达的EGFP融合蛋白的兴趣是在控制的TREtight 诱导启动子。

有了这些工具,就可以通过三个简单的步骤生成用于诱导表达与EGFP融合的目的蛋白的新型细胞模型:

将目标蛋白质的编码序列插入IEX质粒。
将IEX质粒和编码Cre 重组酶的质粒瞬时共转染到配备有一个或两个FC的亲本细胞中。
通过嘌呤霉素选择或荧光激活细胞分选(FACS)选择稳定的整合克隆。
该系统除了易于操作和提高速度外,还可以将插入的拷贝数限制为两个,分别对应于人类基因组中存在的两个AAVS1 等位基因。该系统还可以对几种蛋白质进行比较分析,因为其设计可确保可以并排生成编码不同目标蛋白的细胞,不需要任何特殊资源,除了人cDNA文库和用于扩增并将给定编码序列插入IEX盒中的相应引物外。 EGFP感兴趣的蛋白的编码序列不仅提供了进行活细胞成像的能力,而且EGFP标签还允许通过GFP 纳米阱技术对融合蛋白进行高亲和力纯化,以研究蛋白-蛋白相互作用(Rothbauer 等等,2008)。

我们最近将该系统应用于几种人类神经元细胞模型中的TAU蛋白和BAG5的研究,包括ReN VM人类神经祖细胞,这些细胞可以分化为神经细胞和神经胶质细胞的共培养物(Donato et al。,2007)。研究导致我们发现TAU和BAG5的野生型与突变形式之间的蛋白质-蛋白质相互作用之间存在细微的差异(图1 )。本文旨在共享这些资源并促进其适应其他蛋白质的研究感兴趣的



D:\重新格式化\ 2020-3-2 \ 1902994--1381 Gerold Schmitt-Ulms 846733 \图jpg \图1 rev.jpg

1.设计图中AAVS1 嵌入式系统对于诱导型表达蛋白质的感兴趣。阿。CRISPR-Cas9 切口酶介导的插入基金会纸盒(FC)向AAVS1 安全港。乙编码序列的感兴趣至可诱导中。插入。表达质粒(IEX)ç 。反向四环素控制的反式激活(RTTA 3 )在添加多西环素细胞培养基-介导的基因表达蛋白的感兴趣中。d 实施例1 :.并行诱导的野生型和P301L突变体TAU融合到C-末端EGFP在的ReN ..细胞的抗体针对β-肌动蛋白充当上样对照ë 实施例2:侧由端感应N-末端EGFP -标记的野生型或突变体(DARA)BAG5表达EGFP的细胞仅在此分析中用作其他对照。

关键字:人神经元细胞, CRISPR-Cas9, AAVS1, 诱导表达, 目标蛋白, 增强绿色荧光蛋白

材料和试剂


 


生物材料
质粒(表1)
细胞系(表2)
 


表1. 用于产生诱导性细胞的可用质粒列表及其Addgene ID


Addgene ID


质粒


132388


AAVS1 Kan R 基础熏氧盒


132389


AAVS1 CAG rtTA 3 Tau WT 2N4R-EGFP


132393


AAVS1 CAG rtTA 3 Tau P301L 2N4R-EGFP


132394


AAVS1内含子1 gRNA as2


132395


AAVS1内含子1 gRNA s3


132396


AAVS1内含子1 gRNA as2


 


表2. 可用细胞系列表


细胞系


 


基础熏衣草,HEK293


AAVS1 lox / lox ,转基因插入纯合子,Kan R 片段完整


基础熏鲑鱼IMR-32


AAVS1 WT / lox ,杂合子插入转基因,Kan R 片段完整


基础lox,ReN VM


AAVS1 lox / lox ,用于转基因插入的纯合子,通过优化的Flippase (Flp o )去除了Kan R 片段


诱导型3RWT / 4RWT Tau-EGFP,IMR-32


4R WT Tau-EGFP等位基因:Puro R 被Flp o 移除; rtTA 3 完整


3R WT Tau-EGFP等位基因:Puro R 完整;去除了rtTA 3


诱导型3RWT / 4RP301L Tau-EGFP,IMR-32


4R P301L Tau-EGFP等位基因:Puro R 被Flp o 移除; rtTA 3 完整


3R WT Tau-EGFP等位基因:Puro R 完整;去除了rtTA 3


 


克隆化
注意:所有克隆酶和PCR试剂均在-20°C下保存,稳定1年。


Q5 ® 热启动高保真2 X 预混(新英格兰生物实验室,目录号:M0494S)
底漆:订购为Value Oligos或Custom Oligos ,25 nmole ,脱盐(Invitrogen)。用水稀释至100μM 储备液,在-20°C下储存
Q5 ® 定点突变试剂盒(新英格兰生物实验室,目录号:E0554S )
EZ-10离心柱PCR产物纯化试剂盒(BioBasic ,目录号:BS664-250preps)
带CutSmart缓冲液的限制酶BbvCI (新英格兰生物实验室,目录号:R0601S)
带CutSmart缓冲液的限制性酶NheI -HF(New England Biolabs,CutSmart,目录号:R3131S)
快速AP热敏碱性磷酸酶(Thermo Fisher Scientific,目录号:EF0651)
PureLink 快速凝胶提取试剂盒(Invitrogen,目录号:K210012)
T4 DNA连接酶(新英格兰生物实验室,目录号:M0202S)
的Endura TM 化学感受态大肠杆菌细胞(Lucigen ,目录号:60241-2)店铺在。
-80℃
10自旋EZ柱质粒DNA 小量制备试剂盒(BioBasic ,目录号:BS614-250preps)
的PureLink ® HiPure 质粒筛选马克西试剂盒(Invitrogen,目录号:K210026)
氨苄西林钠盐粉,91.0-100.5%,经过细胞培养测试(Sigma-Aldrich,目录号:A1066-5G)。在4°C下保存,稳定1 年
 


诱导细胞生成
paavCAG-iCre 质粒(Addgene ,目录号:51904,Jinhyn Kim 的礼物)
jetPRIME (PolyPlus ,目录号:114-07).S在4°C时撕裂,稳定1 年
TransfeX 转染试剂(ATCC,目录号:ACS-4005).S在4°C撕裂,稳定1 年
新型Millex -HA注射式过滤器,0.45 Myuemu 孔径(Millipore公司,目录号:5010-SLHA033SS)
新型Millex -GP注射式过滤器,0.22 Myuemu 孔径(Millipore公司,目录号:SLGP033RS)
6孔组织培养板(Falcon,目录号:353046)
24孔组织培养板(Falcon,目录号:353047)
嘌呤霉素(Sigma-Aldrich,目录号:P7255-25MG)。调配至10 mg / ml储备液,过滤灭菌,等分,在-20°C储存
强力霉素(BioBasic ,目录号:DB0889-25G)。重新配制为10 mg / ml的储备液,等分,在-20°C下储存
 


GFP 纳米阱进行亲和捕获
15厘米组织培养板(Sarstedt ,目录号:83.3903 )。
琼脂糖偶联的GFP 纳米捕集阱(Chromotek ,目录号:gta -10)。在4°C下保存,稳定1 年
 


细胞培养
C 6 米组织培养板(Sarstedt的,目录号码:83.3901)
1.5 ml 离心管(Sartsted t ,目录号:72.690.300)
那么-12 组织培养板(萨尔斯塔特,目录号:83.3921)
Dulbecco的磷酸盐缓冲盐水(D-PBS),不含氯化钙和氯化镁,液体磷酸盐缓冲盐水(Sigma-Aldrich,目录号:D8537-500ML)。储存于4°C,稳定1 年
含EDTA 4Na的0.25%胰蛋白酶(Gibco,目录号:25200072)。等分试样,-20°C长期保存。4°C短期保存,稳定6 个月
的StemPro ® 的Accutase ® 。细胞解离试剂(GIBCO,目录编号:A1110501)分装,储存在-20°C的长期储存储存在4℃下短期使用,稳定6个月
基质胶,生长因子降低(Corning,目录号:354230)。等分试样,储存在-20°C,稳定1 年
Recovery TM 细胞培养冷冻培养基(Gibco,目录号:12648010 )。等分试样,在-20°C下储存,稳定1 年
具有高葡萄糖,L-谷氨酰胺和丙酮酸钠的DMEM(Gibco,目录号:11995073)。可在4 °C下保存,稳定1年
合格的10%胎牛血清(Gibco,目录号:12483020)。分装后,可在-20°C长期保存
%1 的GlutaMAX TM -I补充剂(Gibco公司,目录号的:35050061 )。储存在4℃下,稳定1年
DMEM / F12,1:1,包含L-谷氨酰胺,但不含HEPES(Gibco,目录号:21041025)。在4 °C下储存,可稳定1年
2%N21-MAX补充剂(R&D Systems,目录号AR008)或2%B-27无血清补充剂(Gibco,目录号:17504044)。储存在-20 °C下,稳定1年
成纤维细胞生长因子基本(GIBCO,目录号:PHG0261)。存储在-80 ℃下,稳定1叶氩
表皮生长因子(Reprokine ,目录号:RKP01133)。储存于-80 °C ,稳定1年
肝素(Sigma-Aldrich,目录号:H3149-10KU)。储存于-80 °C ,稳定1年
2 ng / ml胶质细胞源性神经营养因子(Gibco,目录号:PHC7045)
500 MYU 中号丁酰-环-磷酸腺苷(Sigma-Aldrich公司,目录号:D0627-250MG)
IMR-32和HEK-293细胞的增殖培养基(请参阅食谱)
ReN VM单元的增殖介质(请参阅食谱)
ReN VM细胞的神经元分化培养基(请参阅食谱)
 


细胞裂解
注:Ø .. Riginal试剂存放在室温下复原库存保存在4℃,持续3个月,所有缓冲器(见食谱)准备从新鲜试剂股票立即使用之前。


Tris-HCl,> 99%(BioShop ,目录号:TRS002.5),用水稀释至1 M
NP-40(BioShop ,目录号:NON505.500)。用水稀释至10%
脱氧胆酸钠(DOC),> 99%(BioShop ,目录号:DCA333.50)。重新配制为pH 8.0的10%w / v的水
氯化钠(NaCl),> 99%(BioShop ,目录号:SOD002.205)。用水稀释至500万
乙二胺四乙酸盐(EDTA),> 99%(BioShop ,目录号:EDT001.500 )。重新配制为水中的0.5 M储备液
钠原钒酸盐(钠3 VO 4 )(BioShop ,目录号:SOV664.25)。重新构建为0.1M的库存在水 
氟化钠(NaF )(BioShop ,目录号:SFL001.100 )。重新配制为水中的1 M库存
苯甲基磺酰氟(PMSF)(BioShop ,目录号:PMS123.5 )。配制为0.5M 乙醇库存
cOmplete 蛋白酶抑制剂(Roche,目录号:11836170001)
PhosStop 磷酸酶抑制剂(Roche,目录号:4906837001)
HEPES(Bioshop ,目录号:HEP001.500)。用水重新配制为0.1万库存
三氟乙酸(TFA),> 99%,HPLC级(Sigma-Aldrich,目录号:302031-100ML )。
乙腈(ACN),HPLC级(Caledon,目录号:1401-7-40)
裂解缓冲液(请参见食谱)
洗涤缓冲液(请参见食谱)
洗脱缓冲液(请参见配方)
 


配套设备


 


-80 °C冰柜(Thermo Fisher,型号:标准性能超低冰柜)
冷冻微型离心机(Eppendorf,型号:5424R,目录号:5404000138)
Avanti J-26S系列高速离心机(贝克曼库尔特,目录号:B22984)
Ø Rbital振荡器(JEIO 技术,型号:ISF-7000系列)
˚F Luorescence显微镜(Leica Microsystems公司,型号:徕卡DMI 6000乙)
NanoDrop 分光光度计(Thermo Fischer Scientific,目录号:ND-1000 )。
ESBE Scientific Save细胞紫外线细胞培养培养箱(Panasonic,目录号:MCO-19AIC)
SterilGARD 生物安全柜(贝克公司,目录号:SG603A-HE)
 


软体类


 


Snapgene(GSL Biotech LLC,https: //www.snapgene.com/ )
Snapgene Viewer(GSL Biotech LLC,https: //www.snapgene.com/snapgene-viewer/ )
Tm计算器(新英格兰生物实验室,https: //tmcalculator.neb.com/#!/ main )。
NEBioCalculator (New England Biolabs ,https: //nebiocalculator.neb.com/#!/ligation )。
NanoDrop 1000 (Thermo Fischer Scientific,https://www.thermofisher.com/ca/en/home/industrial/spectroscopy-elemental-isotope-analysis/molecular-spectroscopy/ultraviolet-visible-visible-visible-spectrophotometry-uv-vis-vis /uv-vis-vis-instruments/nanodrop-microvolume-spectrophotometers/nanodrop-software-download.html)。
程序


 


该协议旨在在不到一个月的时间内(图2 )生成神经细胞模型以进行目标蛋白质的功能分析。该系统可通过程序并行化进行扩展。


 


D:\重新格式化\ 2020-3-2 \ 1902994--1381 Gerold Schmitt-Ulms 846733 \图jpg \图2 rev.jpg


图2.该协议中描述的程序的执行时间表。协议中描述的所有步骤都需要大约一个月的时间来实现。编码可诱导表达的目的蛋白的载体的生成大约可以完成如果选择并验证了阳性克隆,则可以并行进行基于细胞的分析和生化分析。如果计划进行复杂的后续下游分析,则自然会超过一个月的时间表。


 


第一周。将目的基因克隆到IEX
注意事项:


可以使用Snapgene 和Snapgene Viewer 模拟,验证和可视化克隆策略。
可以使用独特的限制性酶切位点BbvCI 和KpnI 将新的目标蛋白的编码序列(CDS)克隆到IEX中:
BbvCI :位于新CDS起始密码子的5' 。


KpnI :存在于将EGFP连接到新蛋白质C末端的接头区域中。


其他独特的限制酶多点(如,NheI位,BstBI位,KflI ,等等。)也可以使用。
 


订购引物,分别将BbvCI 和KpnI 限制性酶切位点附加到目标蛋白质CDS的5'和3'末端。
范例:


引物名称序列             


插入BbvCI 网站5 ' ... CC V TCAGC ... 3' 5 ' ñ 8 CCTCAGC GCCACC ATG X 20-25 3 '             


插入KpnI位网站5 ' ... GGTA V CC ... 3' 5 ' ñ 8 GGTACCX 20-25 3 '             


N 8 代表8个随机核苷酸,用作限制性酶有效消化所需的突出端。
X 20-25 代表目标蛋白质的CDS序列。
蓝色:翻译需要的Kozak序列,包括ATG 起始密码子。
如果新蛋白要表达为EGFP融合蛋白,请不要在用于设计“插入KpnI ”引物的X 20-25 序列中包含终止密码子。
遏制煤矿退火温度引物使用新英格兰生物实验室ŧ 中号计算器工具,它返回此信息在引物序列进入了在线表格字段在:Https://Tmcalculator.Neb.Com/#!/Main。
 


第一天。克隆


使用以下PCR反应执行PCR将BbvCI 和KpnI位点附加到目标蛋白的CDS上:
试剂50 Myueru 反应(Myueru)终浓度                           


Q5 HiFi热启动预混(2x)25 1x                           


模板DNA(20 ng)计算                           


'插入BbvCI '引物(10μM )2.5 0.5μM                           


'插入KpnI '引物(10μM )2.5 0.5μM                           


无核酸酶H 2 O 添加到50                           


总计50                           


执行以下PCR循环:
              步进温度(°C )时间                           


              初始变性98 30 s                           


              变性98 10 s                           


30个循环退火待定30 s                                         


              扩展72 10-20 s / kb                           


              最后延期72 2分钟                           


              保持4 直到样品存储                           


继续按照制造商的规程进行PCR纯化。按照制造商的规程在NanoDrop 1000 上测量DNA浓度。
使用以下反应设置IEX载体和新CDS插入的限制性酶切图:
试剂IEX载体新的CDS插入                           


BbvCI 4微升4微升                           


KpnI 4微升4微升                           


CutSmart缓冲液(10x)5 µl 5 µl                           


碱性磷酸酶1 Myueru -                           


DNA 最高5 µg 最高5 µg                           


无核酸酶的H 2 O 添加至50 µl 添加至50 µl                           


总计50 µl 50 µl                           


在37 °C 孵育1 h。
在孵育过程中,浇铸1%的琼脂糖凝胶以分离消化的产物。
用限制酶切消化混合物的全部体积进行凝胶电泳。
疑难解答:建议将未切割和单切割的IEX向量样本作为对照。


删除双重消化的IEX载体(7.0 kb)和新的CDS插入片段。
按照制造商的规程进行凝胶提取纯化,测量DNA浓度。
 


第1天。将新的CDS插入物连接到双重消化的IEX载体中


确定合适的向量:插入质量比以达到大约1:1的摩尔比,可以使用在线算法来简化此任务,包括NEBioCalculator 工具(1.10.0版; https://nebiocalculator.neb.com/#! /结扎)。
设置以下连接反应:
试剂仅限载体对照(µl)载体+插入(µl)                           


T4 DNA连接缓冲液(10x)2 2                           


T4 DNA连接酶2 2                           


矢量(100 ng)计算计算                           


插入(X伍,取决于比)- 计算                           


无核酸酶H 2 O 添加到20 添加到20                           


总计20 20                           


在室温下孵育15分钟。
按照制造商的规程进行细菌转化。
将琼脂平板上的细菌以100 µg / ml氨苄西林(对于涉及氨苄青霉素的所有应用使用相同的浓度)置于琼脂平板上,将平板在30 °C 下倒置孵育过夜。
Endura具有化学感受态的细胞可在琼脂生长温度为30 °C的情况下最大程度地减少复杂质粒的截短。琼脂平板可减少倒置,以减少过夜孵育过程中琼脂的水分蒸发。


 


第2天。选择转化菌落


比较“仅矢量”控制板上的菌落数(应该没有或只有很少的菌落)和“矢量加插入物”板上的菌落数应该有很多菌落。
6-10菌落从这里选择的“载体加插入”板要生长在3mL的LB培养基氨苄青霉素摇动以225rpm过夜37 ℃下。
 


第3天。提取DNA进行测序


将500 µl过夜细菌培养物保存为甘油储备液。
按照制造商的规程,通过微量制备方法从剩余的细菌培养物中提取DNA。
测量DNA浓度,然后将纯化的DNA质粒送去测序。
其他网站,如果定向诱变要求,进行使用的反应Q5 ® 定点突变试剂盒根据制造商的协议。
 


第4-5 天,Maxiprep 用于转染的新IEX和iCre 载体


使用具有正确测序结果的细菌储备液,使其在含有氨苄青霉素的125-250 ml培养物中生长,然后按照制造商的说明进行DNA maxiprep。
注:这是林Portant使用大量制备试剂盒,去除内毒素从细菌中,以尽量减少不需要的细胞毒性在未来的转染。


同时在,大量制备的ICRE 向量未来的转染。
 


第2周。使用新的IEX载体生成诱导型细胞
通过将IEX载体和iCre 载体共转染,然后进行嘌呤霉素选择和强力霉素(Dox)处理确认可诱导性,设计了下一步步骤以创建可诱导细胞。


 


提前8天


在37度的细胞培养培养箱中培养靶细胞 °C,95%水分和5%CO 2 。
在经过认证的生物安全柜中执行涉及哺乳动物细胞的所有方案,以防止污染并遵守生物安全规则和规定。
转染前至少经过一次以检查细胞是否正常生长,因为融化后它们可能非常脆弱。请参阅“辅助信息”。–“细胞培养”部分,提供有关常规细胞培养,传代和保存库存的说明。
确定合适的嘌呤霉素的浓度,用于进行选择对于每种细胞系通过处理细胞以不同浓度(例如,0.0,0.2,0.5,1.0,2.0,5.0和10 Myug / ml)的中嘌呤霉素以及选择最低浓度杀死100 3天的细胞百分比。
 


第8天。平板细胞进行转染


将所有细胞接种在不含抗生素或抗真菌药的培养基中。
对于ReN VM细胞,在没有肝素的增殖培养基中,在基质胶包被的12孔板上接种约135,000个细胞/孔,第二天应将细胞融合至约50%。
对于不需要铺在带涂层细胞培养皿上的细胞,可在6孔板上每孔接种约350,000个细胞,并期望第二天约80%融合。
保留1-2孔作为有效嘌呤霉素选择的对照;它们不会被转染。
 


第9天。转染


使用1 iCre 载体:4个具有所需插入片段的IEX载体的比例转染细胞。
比例为1 ICRE 矢量:1 - 3 IEX载体还努力。


对于ReN VM单元,总共使用1.0 按照制造商的说明,每孔加入1 g TransfeX 转染试剂和1 g微克 DNA 。
转染过程中应使用不含肝素的增殖培养基,因为肝素可能会影响功效。


对于大多数其他细胞,按照制造商的说明,每孔总共使用3 µg DNA和jetPrime 转染试剂。
对于IMR-32 细胞,请使用1 µg DNA:3 µl jetPrime的比例。


对于HEK-293 细胞,请使用1 µg DNA:2 µl jetPrime的比例。


为了使毒性最小化,在转染到常规增殖培养基后4 h更换培养基。
 


第3周。嘌呤霉素的选择和Dox治疗
将转染的细胞传到一些较大的平板上,并加入适量的嘌呤霉素和2.0 µg / ml的Dox。
一个人可以在荧光显微镜上添加Dox后24小时检查诱导水平。


继续选择嘌呤霉素,直到对照孔中的所有细胞死亡。
注意:建议每隔两天刷新一次嘌呤霉素和Dox,以保持可诱导的转基因转录活性。


允许存活的细胞增殖至大于100个细胞/ 菌落的较大菌落。
用2.0 µg / ml Dox诱导细胞;在荧光显微镜上检查是否成功诱导了集落。
注意:我Nduction在的ReN 。VM细胞可能并非均质即使在同一克隆可能会失去诱导过程中当然分化,这是因为内生后生沉默可能导致变和马赛克诱导型构建(昌达等人,2017年)同样,如果需要分化,可以通过在分化开始之前的增殖阶段2-4天开始诱导来改善ReN VM细胞和其他神经祖细胞的诱导。


标记并以期望的诱导水平将选择的菌落转移至新孔中,将其扩增以保存为原种,并通过蛋白质印迹法验证目的蛋白的诱导性和身份。
去除如果普罗[R 基因是需要的,转染细胞用“ 的FIp Ø ”优化翻转载体。
通过铺平相同细胞克隆的复制品来选择失去嘌呤霉素抗性的细胞,其中一个用嘌呤霉素处理,而另一个则未处理。
在ReN VM细胞中,保留Puro R 可能有助于保持可诱导的转基因区域的转录活性。


 


第4周。为基于质谱的相互作用组研究准备诱导细胞
注意:以下步骤旨在使用GFP 纳米捕集器捕获EGFP标记的诱导蛋白及其相互作用物,以便在质谱仪中进行下游分析。


在15厘米平板上一式三份地培养诱导型目的蛋白EGFP融合细胞,以及诱导型表达EGFP的细胞,仅作为对照,以对抗非特异性结合至GFP nanotrap 基质。
注意:如果诱导表达非常低,请相应放大。


诱导细胞所需的时间长度。
按照制造商的协议使用GFP 纳米阱进行亲和力捕获实验。
所有步骤均应在4°C 下进行,以防止蛋白质降解。


可以按照制造商的规定在RIPA缓冲液中裂解细胞,或将其溶解于0.5%NP-40和0.25%DOC组成的缓冲液中(配方4 )。
通过在4°C下以3,000 xg离心5分钟去除细胞碎片。


由于其高结合力,因此20 µl GFP 纳米阱微珠浆液/生物复制品足以进行基于质谱的相互作用组研究。
EGFP融合蛋白或单独的EGFP可以在与裂解液连续混合,在4°C下孵育2 h的过程中捕获在GFP nanotrap 珠上。
较短的纳阱珠子孵育5分钟,然后轻微离心可能足以可视化EGFP融合蛋白的捕获。


在4°C下以100 xg离心,以收集琼脂糖偶联的GFP 纳米陷阱珠和诱饵蛋白。
在洗涤缓冲液中洗涤磁珠5次,洗涤程序应在2小时内完成,以最大程度减少相互作用物的损失。
在洗涤缓冲液中前3次(配方5 )在这些洗涤步骤中可以使用150-500 mM的NaCl盐浓度,具体取决于所需的严格性。
最后两次洗涤应为25 mM HEPES(pH 7.0)和10 mM HEPES(pH 7.0),不得使用Tris-HCl。
用0.2%三氟乙酸和20%乙腈的水溶液(pH 1.9)洗脱,立即保存并快速冷冻或用pH中和部分洗脱液(例如,总量的25%),以通过Western blot在下游验证诱饵蛋白复合物。
专门用于蛋白质印迹分析的洗脱液收集的延迟可能导致洗脱液蛋白质在pH 1.9环境中沉淀,从而损害了蛋白质印迹分析。


其他特殊注意事项:
可以在亲和力捕获之前使用BCA调整蛋白质含量。
请记住,将一部分细胞裂解液保存为“输入”和“未结合”以进行蛋白质印迹分析。
 


辅助信息


细胞培养


在37 °C 解冻细胞后,通过将细胞原液稀释在4 ml增殖培养基中并在25 °C下以100 xg离心3分钟,从细胞原液中除去DMSO含量。除去淀粉,仅保留细胞沉淀。 DMSO对常规培养的细胞可能有毒。
在适当的增殖培养基中培养细胞约一周,以使其从解冻中恢复。
对于ReN VM细胞,不要在增殖培养基中包含抗生素或抗真菌药,因为它们会导致细胞死亡。
对于ReN VM细胞,在将细胞播种到平板上之前,请至少按照2小时的时间按照制造商的规程用Matrigel涂布组织培养板,Matrigel 在潮湿的培养箱中于37 °C 固化需要2小时。
在细胞达到90%融合之前定期进行传代,以防止使用适当的解离试剂过度生长的不良影响。对于ReN VM细胞,请使用Accutase。对于其他大多数细胞类型,请使用胰蛋白酶。
切记将一部分细胞,特别是有价值的克隆保存为原种,同时将其余的用于实验或常规培养。
为了保存细胞原液,将细胞重悬于标记的冷冻管中的细胞冷冻培养基中,将细胞立即转移至-80°C,并在-80 °C下保存1天,然后再转移至液氮中进行长期保存。
 


资料分析


 


描述该系统首次使用的文章中包含了有关统计分析的信息以及可以从TAU和BAG5分析中找到可公开获得的质谱数据的信息(Wang 等人,2019),其中原始文章的图2提供了定量相互作用组分析设计的详细信息,图3描述了TAU蛋白的质谱序列覆盖范围,图6比较了IMR32和ReN VM细胞中的TAU相互作用组,图9记录了产生的TAU的翻译后修饰分析结果这个系统。


 


菜谱


 


IMR-32和HEK-293细胞的增殖培养基
注意:使介质新鲜,在4°C下储存,稳定2个月。


具有高葡萄糖,L-谷氨酰胺和丙酮酸钠的DMEM培养基,补充了10%合格的牛血清和1%GlutaMAX TM -I补充剂


ReN VM细胞的增殖介质
注意:使介质新鲜,保存在4°C,稳定2周。


DMEM / F12,1:1含L-谷氨酰胺,但不添加HEPES,补充2%N21-MAX或2%B-27无血清


20 ng / ml碱性纤维瘤最后生长因子


20 ng / ml表皮生长因子


2 ng / ml肝素


ReN VM细胞的神经元分化培养基
注意:使介质新鲜,在4°C下储存,稳定1个月。


DMEM / F12 1:1含L-谷氨酰胺,但不添加HEPES并补充2%N21-MAX补充剂或2%B-27无血清补充剂


2 ng / ml神经胶质来源的神经营养因子


500 MYU 中号二丁酰- 环-磷酸腺苷


裂解缓冲液
注:中号阿克鲜用之前,稳定在4 ℃下1天。


0.5%混合物(体积/体积)NP -40,0.5%(W / V)DOC,TRIS / HCl,pH值8.3,5mM的EDTA,1 毫摩尔的Na 3 VO 4 ,10 mM的氟化钠,1 mM的PMSF,    1× 完全蛋白酶抑制剂和1x PhosStop 磷酸酶抑制剂
PR Epare NP-40和DOC作为50X储备溶液和温水轻轻地溶解。DOC会沉淀在PH值低于8.0。因此,确保PH超过此值,并Buffere d添加这种去污剂之前
如果打算使用裂解物进行目标蛋白的免疫亲和捕获,建议在加入蛋白酶和磷酸酶之前准备大量的这种缓冲液,并转移其中一些用于洗涤液(见下文)另外,我们建议在添加温度不稳定的蛋白酶和磷酸酶抑制剂混合物之前,将缓冲液冷却至4°C。


洗涤缓冲液
注意:使用前应使其新鲜,并在4 °C下稳定2天。


考虑以下因素,该缓冲液的组成与裂解缓冲液相似:


蛋白酶和磷酸酶抑制剂可以省略。


如果样品在下游用于质谱分析,则最后两次洗涤必须仅在去离子水中使用25 mM HEPES(pH 7.0)和10 mM HEPES(pH 7.0),不得使用Tris -HCl。 


洗脱缓冲液
注意:使用前应使其新鲜,在室温下稳定1天。


在去离子水中混合0.2%的TFA和20%的乙腈
此溶液的pH值为1.9,无需调整
 


致谢


 


XW持有安大略省研究生奖学金,GS 获得了克里姆比尔基金会,加拿大卫生研究所(授权号137651)和艾伯塔省Prion研究所(授权号201600028)的资金。阿诺德·欧文家族的支持下购买了质谱仪。对此项目的启动起了重要作用。作者深表感谢Borden Rosiak 家人的慷慨慈善支持。


 


竞争利益


 


作者宣称他们没有金融或非金融竞争利益。


 


道德规范


 


该研究不涉及人类受试者或动物工作,人类细胞系和化学物质的所有处理均根据大学环境健康与安全(EHS)办公室批准的有效生物安全规程(编号208-S06-2)进行多伦多,加拿大多伦多市。


 


参考文献


 


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引用:Wang, X., Friesen, E., Müller, I., Lemieux, M., Dukart, R., Maia, I. B., Kalia, S. and Schmitt-Ulms, G. (2020). Rapid Generation of Human Neuronal Cell Models Enabling Inducible Expression of Proteins-of-interest for Functional Studies. Bio-protocol 10(9): e3615. DOI: 10.21769/BioProtoc.3615.
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