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

Abstract

Our group has developed several approaches for stable, non-viral integration of inducible transgenic elements into the genome of mammalian cells. Specifically, a piggyBac tetracycline inducible Genetic Elements of Interest (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 doxycycline (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. More recently, we’ve developed an alternative transgenic system to piggyBac called Mosaic Analysis by Dual Recombinase-mediated cassette exchange (MADR), as well as additional in vitro transfection techniques and in vivo doxycycline chow applications. The protocols herein provide instructions for the use of this system in cell lines and in the neonatal mouse brain.

INTRODUCTION

This article describes protocols for use of the Tet inducible “genes of interest” (GOI) systems using piggyBac and Mosaic Analysis by Dual Recombinase-mediated cassette exchange (MADR) transgenic technologies, which allow for flexibility in terms of the expression of genetic elements, including transgenes, shRNAs, or sgRNAs (Akhtar et al., 2015; Kim et al., 2019). Specifically, there are several different tetracycline transactivators to allow for doxycycline (dox)-mediated suppression (tTA), activation (rtTA-V10), constitutive expression (tTA H100Y), or induced constitutive expression (rtTA-V10 + inducible tTA H100Y) (Akhtar et al., 2015). The value of such control over transgenes derives from the fact that many genes are “recycled” throughout organs and tissues for different uses during development, adulthood, and in disease (Ables, Breunig, Eisch, & Rakic, 2011). Additionally, many genes—such as those involved in cellular reprogramming—may cause deleterious effects if misexpressed over protracted periods (Akhtar & Breunig, 2015). For these reasons, investigation of gene function in these diverse contexts requires increasingly precise control over transgene expression. Furthermore, plasmids using piggyBac transpositions (pB) as a means of genomic integration of transgenic elements, such as a Tet-On inducible GOI system (Tet-GOI), allows for stable transduction of cell lineages through non-viral means (Breunig et al., 2015; Chen & LoTurco, 2012). A novel alternative transgenic technique to piggyBac called MADR has been developed as an additional technology to stably transfect mammalian cells (Kim et al., 2019). MADR is a non-viral transgenic technique which has advantages over the piggyBac system in that it stably integrates donor plasmids (pDonor) between recombinase sites; both controlling for the site of insertion and number of integrations.

Both the transgenic systems, pB and MADR, discussed in this protocol are compatible with in vitro and in vivo models. Various cell types can be transfected in vitro including iPSCs, iPSC-derived neural progenitors, primary mouse neural progenitors, and human HEK293 cells, among others. In vivo models can be generated by transplanting transgenic cell lines into a mouse or by directly transfecting progenitors in the brain using electroporation. Extensive testing of postnatal electroporation of postnatal 2–3 day old (p2-p3) mouse pups has been performed to transfect radial glia progenitors lining the ventricle and results in differentiated daughter cells which contain the transfected plasmids (Kim et al., 2019). Different cell populations may be targeted with in vivo electroporation based on the temporal and spatial parameters, as well as by using cell specific promoters in the inducible plasmids. In vivo imaging of the transfected cells is possible using luciferase to assess transgenic gene expression in a non-invasive manner.

Herein, we provide a detailed protocol for the use of transgenic systems in vitro and in vivo in order to inducibly control gene expression. Basic Protocol 1 details how to clone a plasmid that will control the expression of genetic elements of interest using one of the three different Tet transactivator protein. Two different possible transgenic systems will be discussed, the piggyBac transposon system and MADR transgenesis system. Basic Protocol 2 details how to create stable cell lines in vitro containing the cloned plasmid by nucleofecting either mouse or human iPSC derived neural progenitors. Basic Protocol 3 discusses how to induce the expression of GOI both in vitro and in vivo using doxycycline. Basic Protocol 4 describes a bioluminescence detection system to assess the induced gene expression in vivo.

STRATEGIC PLANNING

Recent alternative approaches for this protocol include the use of a novel transgenic technology Mosaic Analysis by Dual Recombinase-mediated cassette exchange, or MADR (Kim et al., 2019). MADR is a non-viral technique developed in-house which relies upon two recombinase enzymes, Cre and FlpO, to undergo recombinase mediated cassette exchange (RMCE) between LoxP and FRT sites (Fig. 1C). This ensures a single copy of the donor plasmid (pDonor) is stably integrated in a controlled location, avoiding off target insertion-deletion mutations (InDels) that are observed with randomly integrating transgenic technologies like PiggyBac and viral constructs (Schröder et al., 2002). MADR is ideal for studying genes whose function changes based on gene dosage or when a physiological normal gene expression level is desired, such as dosage-sensitive developmental processes (i.e., the Notch pathway) or oncogenesis (for discussion see Kim et al., 2019). MADR has been shown to be compatible with the Tet inducible system in which each cell can only integrate a single response plasmid at one time (Kim et al., 2019).

Figure 1.

The Tet-GOI systems; piggyBac (A) and MADR (C) transgenic systems. (A1) pCAG-pBase. piggyBac transposase (pBase) plasmid which constitutively expresses pBase protein under the pCAG promoter. (A2) pB-rtTA-v10. The piggyBac transactivator plasmid which constitutively expresses the rtTA-V10 transactivator. (A3) pB-TRE-Bi-clover-luciferase/GOI. Response plasmid which constitutively expresses TagBFP2-V5-nls (Blue fluorescence protein (BFP) with a V5 tag and nuclear localization sequence (nls)) and inducibly expresses membrane clover/luciferase along with a GOI (genetic element of interest) under the bi-directional (Bi) Tet response element (TRE) (TRE-Bi). (B1-B2) Cartoon demonstrating effect of addition of doxycycline (dox) to the cells harboring pB-tet-GOI system, using the Tet-on transactivator. (C1) pCag-FlpO-2A-Cre. Dual recombinase plasmid containing both Cre and FlpO recombinases under the pCag promoter, which is used to perform recombinase mediated cassette exchange with MADR. (C2) MADR compatible Tet-on inducible plasmid containing both the transactivator (left segment) and response plasmid (right segment) in one. This plasmid has a constitutively active mScarlet reporter and an inducible “spaghetti monster” fluorescent protein (SMFP)-EGFP-Myc reporter (Viswanathan et al. 2015). Note the LoxP and FRT sites in place of the PB TR. (C3) MADR integration site in the Rosa26 locus in the mTmG mouse. Cre and FlpO recombinases allow for cassette swapping between LoxP and FRT sites. (D1-D2) Cartoon demonstrating the effect of dox on cells transfected with MADR-tet-GOI system; transfected cells express constitutive active mScarlet fluorescent reporter and the addition of dox induces the SMFP-EGFP-Myc fluorescent reporter as well as the downstream GOI.

Each transgenic technology has advantages and disadvantages which should be considered when planning an experiment. While MADR controls for insertion location and copy number, piggyBac has the advantage of an increased transfection efficiency and can result in increased gene expression due to multiple insertions. This would be advantageous in situations of using fluorescent reporters to identify specific cells of interest.

The cloning preparation for both MADR and piggyBac compatible plasmids is similar, except in MADR the donor plasmid is flanked by LoxP and FRT sites, while piggyBac inserts are flanked by piggyBac terminal repeats (PB_TR) (Fig. 1C2). Since MADR only allows a single donor plasmid (pDonor) to integrate in each cell at a time, the transactivator plasmid and response plasmids are combined into one large “donor” plasmid separated by a P2A element (Rincon Fernandez Pacheco et al., 2020).

Instead of using the pBase integrase plasmid with piggyBac, MADR uses a plasmid containing two recombinase enzymes, Cre and FlpO, separated by a P2A element (Fig. 1C1). The pBase and pCag-FlpO-2A-Cre plasmids are episomally expressed, so after the expression of the integrase or recombinases “wash out”, the inserts will be stably integrated. Since MADR pDonor plasmids can only integrate between LoxP and FRT sites, transgenic mTmG mice are typically used as they contain LoxP and FRT sites in the Rosa26 locus (Muzumdar et al., 2007) (Fig. 1C3). Proof of concept using human HEK293 cells has displayed LoxP and FRT sites can be inserted into the AAVS1 locus in human cells to create a human “proxy” line containing LoxP and FRT sites, generating cells which are compatible with MADR (Ayala-Sarmiento et al., 2020; Kim et al., 2019).

In vitro transfection can be performed with two different electroporation devices: the Lonza nucleofector or the MaxCyte electroporator. In the authors experience, both devices can successfully transfect neural precursor cells and should be viewed as comparable techniques. The MaxCyte electroporator specializes in delivering genomic material to sensitive and hard to transfect cells, such as iPSCs. Additionally, the device is highly customizable and can be scaled up to larger quantities of cells all while using the same machine and variables. Lonza nucleofector also can transfect a wide variety of cell types but cannot scale up to large quantities of cells. This protocol can work using either device and a decision of which to use should be based on availability and prior experience with the devices.

For in vivo experiments, in utero and post-natal delivery methods of the Tet-GOI and MADR system are not limited to electroporation of the lateral ventricle, although this technique has been typically employed by the authors (Akhtar et al., 2015; Kim et al., 2019). Timing of electroporation will dictate the cells transduced with in utero electroporation allowing genetic access to embryonically generated populations and postnatal electroporation targeting latent neural precursors and derivative lineages based on cell birthdates (Breunig et al. 2011). Alternative methods of gene delivery to other regions of the brain, such as pial surface electroporation or cloning the response and transactivator plasmids into viral vectors may also lend well to the use of the system (Braun, Machado, & Jessberger, 2013; Breunig et al. 2012; Levy, Molina, Danielpour, & Breunig, 2014). Specifically, therapeutic approaches to the CNS may favor viral-mediated delivery approaches (Chtarto et al., 2016). Additionally, transplantation of cells harboring the Tet system into the CNS may allow for cells to engraft and proliferate before activating GOI activity by administration of dox.

BASIC PROTOCOL 1

CLONING OF RESPECTIVE GOI (GENETIC ELEMENT OF INTEREST) INTO RESPONSE PLASMID

The pB-tet-GOI system involves three plasmids and allows for the flexible expression of a GOI (Fig. 1) (Akhtar et al., 2015). The first plasmid constitutively expresses the pBase integrase enzyme (Fig. 1A1). pBase catalyzes the random stable integration of plasmids that are flanked with piggyBac terminal repeats (PB TR) (Breunig et al., 2015; Chen & LoTurco, 2012). The second plasmid constitutively expresses a transactivator protein; the rtTA-V10 (Tet-On), shown here (Fig. 1A2). The third plasmid, the response plasmid, constitutively expresses nuclear blue fluorescent protein coupled to a V5 protein tag (TagBFP2-V5-nls), allowing cells that have been transfected to be fluorescently identified and isolated by FACS (Fig. 1A3). Upon addition of doxycycline (dox) to the system, the rtTA-V10 transactivator undergoes a conformational change, which allows it to bind the Tet response element bi-directional promoter (TRE-Bi) of the response plasmid (Fig 1A3). This catalyzes inducible expression of any gene of interest as well as the luciferase and clover reporters (Fig. 1A3).

Alternatively, replacing the rtTA-V10 (Tet-On) transactivator plasmid depicted (Fig. 1A2) with one of three other transactivators will yield different results upon the addition of doxycycline. Firstly, the tTA2 (Tet-Off) transactivator allows expression of the GOI and clover/luciferase reporters in the absence of dox, and the addition of dox silences transcription. Alternatively, the tTA2-CA (Tet-insensitive) plasmid allows constitutive expression of GOI and clover/luciferase irrespective of the presence or absence of dox. Lastly, if constitutive expression after a single dose of dox (induced non-reversible) is desired, using the rtTA-V10 (Tet-On) plasmid along with an inducible Tet-insensitive (i-tTA2-CA) transactivator will allow continuous expression after a single dose of dox; this is similar to the effect seen when a cre recombinase is used to excise a stop site upstream to a gene of interest. For the purpose of this protocol, the rtTA-V10 (Tet-On) transactivator will be used for all experiments (Fig. 1A2).

The response plasmid (Fig. 1A3) features multiple unique restriction enzyme sites flanking the “GOI”, allowing genes to be easily replaced. “P2A” elements—ideally codon alternated to avoid repeat nucleotide sequences—can be used to express multiple GOIs in tandem (Fig. 1C1–C2; see Kim et al., 2019 for examples). In addition, the transactivator and response plasmids can be combined into one larger plasmid (Fig. 1C2).

Materials

Use plasmids for either piggyBac (3 plasmids) or MADR (2 plasmids):

pB-Tet-GOI system (three plasmids):

pCAG-pBase: pBase plasmid that constitutively expresses pBase protein (Addgene Plasmid cat. no. 40972).

pCAG-rtTA-v10-pB: A transactivator plasmid that constitutively expresses the rtTA-V10 transactivator (Plasmid available by request).

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) (Plasmid available by request).

MADR-Tet-GOI system (two plasmids):

pCag-FlpO-2a-Cre: Dual recombinase plasmid for the recombinase mediated cassette exchange of the pDonor plasmid between LoxP and FRT sites (Addgene Plasmid cat. no. 129419).

pDonor-rtTAv10-mScarlet-TRE-SMFP-EGFP-Myc-GOI: Transactivator and response plasmid together in a MADR compatible donor plasmid with a constitutive mScarlet fluorescent reporter and an inducible SMFP-EGFP-Myc fluorescent reporter followed by a site for GOI (Plasmid available by request).

Restriction enzymes (New England BioLabs)

Custom DNA Primers (Integrated DNA Technologies)

Nuclease-Free Water (Fisher Scientific, cat. no. AM9937)

KAPA HiFi DNA Polymerase HotStart ReadyMix (Roche Sequencing Store, kit code KK2601)

E-Gel EX 1% Agarose (Fisher Scientific, cat. no. G402021)

E-Gel 1 Kb Plus DNA Ladder (Fisher Scientific, cat. no. 10488090)

NucleoSpin Gel and PCR Clean-up kit (Takara Bio, cat. no. 740609.50)

NEBuilder HiFi DNA Assembly Master Mix (New England BioLabs, cat. no. E2621S)

Stellar Competent Cells (Takara Bio, cat. no. 636763)

One Shot Stbl3 Chemically Competent E. coli (Thermo Scientific, cat. no. C737303)

Carbenicillin Disodium Salt (AG Scientific, sku C-1385-1GM)

14 mL Polypropylene Round-Bottom Tube (Thermo Scientific, cat. no. 14-959-11A)

LB Broth tablets (Sigma-Aldrich, cat. no. L7275-100TAB)

LB Broth 1000mL (Fisher Scientific, cat. no. 50-591-332)

Glycerol (Sigma-Aldrich, cat. no. G5516-100ML)

NucleoSpin Plasmid miniprep kit (Takara Bio, cat. no. 740588.50)

NucleoBond Xtra Maxi Endotoxin-free (Takara Bio, cat. no 740426.50)

Bio-Rad C1000 Touch Thermal Cycler

E-Gel Power Snap Electrophoresis Device (Thermo Scientific, cat. no. G8100)

Eppendorf 5417R Benchtop Lab Centrifuge

Beckman Coulter Avanti JXN-26 High-Speed Centrifuge

Steris Amsco Century SV-1262 Prevac Steam Sterilizer

Protocol steps with step annotations

The following cloning instructions are general guidelines as every cloning project is unique, but the workflow can be similar. Best practice involves starting with a plasmid backbone, digesting at specific sites with restriction enzymes to remove undesired elements, PCR desired elements from template fragments, and combine all the fragments together with NEB assembly. For a much more detailed protocol, please refer to Rincon Fernandez Pacheco et al., 2020.

1. Digest the tetracycline response plasmid with necessary dual restriction enzymes to excise undesired GOI. For the digestion, add 1μg of DNA, 5μl 10X CutSmart Buffer, 1μl of each restriction enzyme, and bring the total volume to 50μl using Nuclease-free Water. Incubate at 37°C for 1hr.

2. PCR GOI from template plasmid using primers that contain a homology overlap of 25bp to the neighboring fragment you will be assembling as per Gibson or NEB HiFi Assembly instructions. (Alternatively, 15 bp overlaps are typical for In-Fusion cloning). For the PCR parameters, use less than 10ng of the template DNA, 1μl of each the forward and reverse primer (10μM stock), 12.5μl of 2x PCR Mastermix, and nuclease-free water to bring the total volume to 25μl.

   Calculate annealing temperature for the primers using only the sequence that binds to the template, excluding the homology overlap segment.

3. Clean up the digestion and PCR fragments by running them on a 1% agarose gel.

4. Cut the band of the desired length out from the gel and clean up using the NucleoSpin Gel and PCR Clean-up kit.

5. Perform a NEB assembly using 10μl of 2x HiFi Assembly Master Mix and 10μl of vector + insert + H20, for a total reaction of 20μl. Run assembly at 50°C for 30min.

   Calculate optimal quantities of DNA fragments and ratios using a molar ratio calculator such as NEBiocalculator.

6. Transform less than 5ng of pure DNA into Stellar Competent Cells. Follow Stellar competent cell transformation protocol. This involves thawing the cells on ice for 30 minutes, heat shock the cells for 45 seconds at 42°C, rest the cells on ice for 1-2min, add SOC medium to bring the total volume to 500μl, and incubate for 1hr at 37°C with shaking (160-225 rpm). Streak an appropriate amount (about 20-50μl, depending on concentration) on an agar plate with the appropriate selection antibiotic. Incubate overnight for about 12-16hr.

   Stbl3 Chemically Competent cells may be used to reduce recombination in large plasmids or plasmids with many repeats. Note Stbl3 cells incubate at 30°C and need additional incubation time.

7. Pick an isolated colony and in 14mL round bottom tubes, create a mini culture in 3mL LB broth with 1:1000 of selection antibiotic. Close lid to the position that allows air exchange, but do not fully seal the lid. Culture overnight at 37°C with shaking (160-225 rpm).

   Recommended to select at least 3 independent colonies for miniprep and sequencing.

8. Next morning, check that mini culture has growth by swirling the tube and observing how cloudy the LB broth is.

9. Create a glycerol stock using 750mL mini culture + 375mL LB broth + 375mL glycerol and store at −80°C.

10. Perform a miniprep with the remaining mini culture to isolate DNA for sequencing using the NucleoSpin Plasmid miniprep kit.

11. Sequence DNA isolated from mini culture with primers covering sites of ligation and PCR.

12. When sequence is confirmed to be correct, move on to an endotoxin-free Maxiprep.

13. The morning of maxiprep, begin a starter culture using 3mL LB broth, 1:1000 of selection antibiotic, and a small amount of the glycerol stock generated in step 9 (about the size of a bacterial colony). Culture at 37°C, 160-225RPM, for ~8hr.

14. Prepare LB for maxiprep by combining 6 LB broth tablets with 300mL distilled water in large Erlenmeyer flask. Cover with foil and autoclave. Let cool after autoclave.

15. Prepare a Maxiprep culture by combining 300mL LB broth + 1:1000 selection antibiotic + 1:1000 of the starter culture prepared in step 13.

16. Follow Maxiprep protocol to obtain a pellet of pure endotoxin-free DNA using the NucleoBond Xtra Maxi Endotoxin-free kit. Highly concentrated stock solutions (i.e., >3 μg/μl) allow flexibility for use in vivo and in vitro.

BASIC PROTOCOL 2

In Vitro Nucleofection of iPSC-Derived Human/Mouse Neural Progenitor Cells and Subsequent Derivation of Stable Inducible Cell Lines

The pB-tet-GOI system can be introduced by various methods into cell lines (lipofectamine-mediated, sonication, nucleofection, etc.). We recommend using methods that have proven to be effective for introducing larger sized plasmids (~10 kb) into the desired cell lines. With this in mind, we provide a detailed protocol below for the nucleofection of human induced pluripotent stem cell-derived neural progenitor cells (iPSC-derived NPCs) (Ebert et al., 2013; Mattis et al., 2015). This protocol has also been successfully used for the nucleofection of human primary neural progenitor cells and mouse neural stem cells (Akhtar et al., 2015; Kim et al., 2019).

MADR has been extensively tested in vitro in a variety of cell lines (HEK293, iPSCs, iPSC-derived NPCs, primary NPCs, etc.) as well as using lipofectamine and nucleofection. The ratio for integration of donor plasmid (pDonor) to recombinase plasmid has been empirically found to be 10:1 (pDonor to pCag-FlpO-2A-Cre) (Kim et al., 2019), although optimization for cell type and plasmids should be performed. The following protocol will continue to use the piggyBac plasmid as an example since the transfection process of MADR and piggyBac plasmids are identical.

Additional adaptations to the in vitro transfection protocol include an optional “recovery step” as well as using the MaxCyte electroporator as an alternative to the Lonza nucleofector. The MaxCyte electroporator has been found as an alternative tool to deliver large plasmids into difficult to transfect cells, such as human iPSCs and NPCs. The optional recovery step after transfection allows for the cells’ plasma membranes to stabilize before being disturbed and has been found to greatly increase transfection viability. Additionally, the use of conditioned growth medium to neutralize the enzymatic disassociation reagents have also been found to increase viability during disassociation.

NOTE: For all the procedures described which involve the culturing of cells, standard tissue culture facilities are required, and sterile culture techniques should be employed. Cultures should be grown at 37°C in an incubator with proper humidity and 5% CO₂. It is important to properly dispose of sharps and contaminated material.

NOTE: Fire polishing glass pipettes requires the use of a flame in a sterile environment. Care should be taken when using an open flame. Glass pipettes should be handled with caution as a sharp edge can occur from broken tips.

Materials

Nucleofection kit (Lonza, cat. no. VPG-1004) containing:

Nucleofection reaction solutions

pBase plasmid, transactivator plasmid, and response plasmid (from Basic Protocol 1, Fig. 1A)

or

MADR pDonor plasmid and dual recombinase plasmid (from Basic Protocol 1, Fig. 1C)

MaxCyte Electroporation kit containing:

MaxCyte ATx electroporator

OC-100x2 cassette (MaxCyte, cat. no. SOC-1x2)

MaxCyte electroporation buffer (MaxCyte, cat. no. EPB-1)

Human iPSC-derived NPCs (Ebert et al., 2013; 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 in Reagents and Solutions)

Cell scraper, optional

Hemacytometer

Disposable cotton-plugged borosilicate-glass Pasteur pipettes (Fisher Scientific, cat. no. 13-678-8B)

70-μm cell strainer (Falcon, cat. no. 08-771-2)

Y-27632 ROCK inhibitor (Tocris Bioscience, cat. no. 1254/1)

Corning Costar Ultra-Low Attachment Microplates (Fisher Scientific, cat. no. 07-200-601)

DNase I Solution (Thermo Scientific, cat. no. 90083)

DMEM (Fisher Scientific, cat. no. 11-965-092)

200ul sterile pipette tips

15mL sterile conical tubes

5mL sterile serological pipettes

1-ml pipettes

CELLstart Substrate (Fisher Scientific, cat. no. A1014201)

Corning Matrigel Growth Factor Reduced Basement Membrane Matrix (Millipore Sigma, cat. no. CLS356230-1EA)

DPBS, No calcium, no Magnesium (Fisher Scientific, cat. no. 14190144)

Lonza Nucleofector 2b device and corresponding solutions/cuvettes (specific to cell type being nucleofected)

Eppendorf Centrifuge 5702

Thermo Scientific HERAcell 150i CO2 37°C incubator

NOTE: The data presented here involves immunostaining or imaging of native fluorescence. For the former, the following antibodies have been verified for staining V5, clover, luciferase, mScarlet, and Myc-epitope 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),

Rabbit anti-luciferase 1:1000 (Abcam, cat. no. 21176),

Rat anti-tdTomato 1:250 (Kerafast, cat. no. EST203), and

Human anti-c-myc epitope tag 1:5000 (Absolute Antibody, cat. no. Ab00100-10).

Protocol steps with step annotations

Nucleofect pB-tet-GOI into human iPSC-derived NPCs

• 1.
  For nucleofection solution composition, follow the Lonza manufacturer’s instruction. Briefly, to each Lonza Nucleofection reaction (which contains 100 μl of Lonza solution; 82ul Solution + 18ul Supplement), add 1 μg of pBase plasmid, 3 μg of transactivator plasmid, and 7 μg of response plasmid. If several response plasmids are being added, add 5 μg of each response plasmid. The total amount of DNA per reaction should be <25 μg and the total volume should not exceed 120 μl. Inducible GOI expression has been observed using up to four response plasmids.

  Note: not all cells will receive all four response plasmids; therefore, inserting transgenes in tandem into one response plasmid using “P2A” linkers or multiple cistrons may be preferred if a more homogeneous population of transfected cells is desired.

• 2.
  Human iPSC-derived NPCs can be grown on a monolayer or in suspension as spheres, each requiring different dissociation techniques.

  Note: Prolonged proliferative capacity has been observed when growing the NPCs in suspension as spheres.

Dissociation of cells grown on monolayer

• 3a.
  If cells are grown on a monolayer, dissociate cells using desired cell dissociation enzyme. TrypLE express is preferred for human iPSC-derived NPCs. Manual dissociation using a cell scraper and no enzyme may also be performed for cells that are not accustomed to enzymatic dissociation. Alternatively, a combination of both enzymatic and mechanical dissociation can be used.

  1. Remove the conditioned growth medium (see recipe in Reagents and Solutions) from flask and save for later. Wash once with 10 ml PBS to remove traces of growth medium.
  2. Add enough TrypLE to cover flask (5 ml for 75-cm² flask) and incubate for 5 min at 37°C or until cells start to lift off monolayer. After 5 minutes of enzymatic dissociation, a wide bore P1000 pipette tip or cell scraper can be used to mechanically disassociate remaining adherent cells.
  3. Collect dissociated cells in a 15-ml conical tube. Neutralize the enzymatic reaction by adding equal or more conditioned growth medium. Using conditioned media to neutralize the enzymatic reaction has been found to increase viability, but fresh media can also be used.
  4. Centrifuge for 5 min at 150 × g, 23°C, to pellet the cells. Count the cells using a hemacytometer. Obtain a pellet of 3 to 5 million cells.

Dissociation of cells grown as spheres

• 3b.
  If cells are grown as spheres, gently dissociate spheres to near single-cell suspension to increase nucleofection efficiency. Clumps of about 3-6 cells can encourage better transfection efficiency and viability than complete single-cell suspension.

  • i
    Fire polish three glass plugged pipette tips in sequential diameter for the purpose of slowly dissociating spheres through a progressively smaller diameter pipette. Ensure ends are smooth and not chipped as sharp ends can shear cells.
  • ii
    Remove cells from the flask and place in a 15-ml conical tube. Allow spheres to settle by gravity (1 to 2 min). If spheres are small and continue to float, a gentle spin (3 min at <125 × g) will promote settling.
  • iii
    After cells have settled, remove, and save the conditioned growth medium and add 4 ml of warm TrypLE. Incubate for 5 min at 37°C.
  • iv
    Add 6 ml warm conditioned or fresh growth medium to neutralize TrypLE reaction. Gently centrifuge (5 min at 150 × g, 23°C). Carefully discard the supernatant using a pipette.

Pellet will likely dislodge easily, so pouring out medium or using a vacuum is not recommended.

• vUsing a 1-ml pipette, add 1 to 2 ml of warm growth medium to pellet and gently pipette up and down five times to manually dissociate spheres. Let non-dissociated spheres settle by gravity flow (1 to 2 min) and SAVE the supernatant, which contains dissociated cells.
• viLubricate the inside of the pulled glass pipette tips by aspirating fresh growth medium in and out of pipette once. This prevents cells from attaching to inside wall of the pipette. Ensure medium does not reach the cotton plug.
• viiUsing the largest diameter fire-polished glass pipette, add 1 to 2 ml of fresh warm medium to pellet. Carefully pipette up and down 5 times to dissociate spheres. Let non-dissociated spheres settle by gravity flow (1 to 2 min) and SAVE the supernatant, which contains dissociated cells. Repeat this step with the remaining smaller diameter fire-polished glass pipettes.
• viiiOptional: Dissociated cell solution can be passed through a 70-μm filter. While this may decrease clumping of cells, depending on the cell type, it may increase stress and reduce cell viability.

• 4.Count cells using a hemacytometer. Gently centrifuge 3 to 5 million cells 5 min at 150 × g, 23°C, to obtain a pellet.
• 5.To the pellet of 3 to 5 million cells, add (all 100 μl) of nucleofection solution containing plasmids (from Basic Protocol 2, step 1) to cell pellet and gently mix to ensure a homogeneous solution. Transfer this solution to a cuvette (provided with Lonza kit).
• 6.
  Place the cuvette in the Lonza nucleofector and select the desired pre-programmed cell type. Push the “OK” button to nucleofect the cells.

  • a
    To increase cell transfection viability, an optional recovery stage can be performed as described below in step 20.
  • b
    Note: observing a white protein like substance after transfection is common when using Lonza nucleofection.
• 7.
  Pre-wet the inside of plastic eyedrop pipette (provided in Lonza kit) with fresh warm medium. After either the optional recovery stage or directly after transfection, transfer the nucleofected cells to 1 ml of warm media.

  If recovery stage is performed, skip to step 20, and then resume at step 8.

• 8.
  Count the cells using a hemacytometer.

  It is common for 30% to 60% of cells to die from the nucleofection.

• 9.
  Dilute/concentrate the cells as desired and plate on desired substrate. If growing as a monolayer, performing a “dry plate down” may increase adherence. If growing as spheres, plating cells at a high concentration will promote sphere formation.

  • a
    For dry plate down: plate the cells down only in enough growth medium to cover the surface of a plate coated with the desired matrix (e.g., 1 to 2 ml for a 25-cm² flask). After 4 to 8 hr, live cells will adhere, and the flask can be flooded with growth medium. For human iPSC-derived NPCs, plates/coverslips coated with Matrigel or CELLstart are preferred.

Matrigel coating:

1. Place P200 tips, sterile 15mL conical tube, and 5ml sterile serological pipette at −80°C for 1hr.

2. The working concentration of Matrigel is 0.5mg Matrigel per 6mL DMEM (for 6-well plate use 1mL/well)

3. Remove items from −80°C freezer and place in a sterile biosafety cabinet.

4. Transfer desire volume of DMEM into cold 15mL conical tube.

5. Using cold pipettes, use cold media to thaw the frozen Matrigel by pipetting up and down. Avoid holding tubes at the level of the Matrigel as the warmth from your fingers can solidify the Matrigel.

6. Transfer thawed Matrigel to 15ml conical tube with cold DMEM and continue to mix with the 10ml serological pipette.

7. Work quickly.

8. Transfer the Matrigel/DMEM mixture to tissue culture dish.

9. Swirl to coat the surface.

10. Incubate at room temperature for 1 hour.

11. If plates are not immediately used, add 1mL additional DMEM to each well and store in 4°C fridge for up to 1 week.

12. Do not allow wells to dry out or they cannot be used.

CELLstart coating:

1. Calculate desired volume of coating matrix (6-well plate = 1mL/well)

2. Transfer DPBS without Calcium and Magnesium to 15ml conical tube.

3. Dilute CELLstart in DPBS at a concentration of 1:50.

4. Transfer diluted CELLstart into culture vessel.

5. Incubate in 37°C incubator for 1hr.

6. Do not allow coating to dry out.

7. Remove CELLstart and replace with media.

• bFor growing human iPSC-derived NPCs as spheres, ultra-low attachment plates may be used. However, for growing as spheres after nucleofection, it is recommended to allow cells to recover from nucleofection by plating down on adherent substrate, growing to confluency, and then transferring to ultra-low attachment plates for sphere formation.

• 10.Treat cells as normally treated before nucleofection. Perform a media change the day after nucleofection to remove dead cells and debris. It is recommended to wait 12-24 hr before adding dox to medium (described in Basic Protocol 3).
• 11.To generate stable cell lines before activation of GFP/GOI, fluorescence-activated cell sorting on the constitutively activated BFP (blue fluorescence protein) can be used. Alternatively, a transactivator plasmid containing puromycin resistance can be used and a stable cell line can be generated by selecting on puromycin and BFP expression. Cells can also be sorted for GFP after adding dox.

[*Copyeditor: The alternative protocol below is missing an introduction section. If the authors want to keep it as the alternative protocol, they should renumber the steps and the protocol, and its title should be listed in the Abstract section. Alternatively, this protocol can be named Alternative Steps and the step numbers be kept as they are. Please check with authors]

Alternate Protocol 2: Transfection protocol using MaxCyte electroporator (Performed in place of steps 5-7 above, resume with plating step 9)

Note: Refer to the MaxCyte manuals and field application specialists for the most up-to-date protocols for transfecting specific cell types. The following are conditions successfully used to transfect human iPSCs before differentiation into NPCs.

• 12.Optional: Passage iPSCs the day before transfection to keep cells in the logarithmic growth phase. Replate cells at 80% confluency and supplement media with 1 μM Y-27632 ROCK inhibitor overnight.
• 13.Dissociate cells as described above in step 3 and spin for 5 min at 150 × g, 23°C, with a low break.
• 14.Remove supernatant and gently resuspend pellet in MaxCyte electroporation buffer at 1:10 volume, using a P1000 tip (wide bore if available) and make a near single-cell suspension.
• 15.Count cells using a hemacytometer.
• 16.Pellet cells again at 150 × g, 23°C, with low break.
• 17.Resuspend pelleted cells in MaxCyte buffer to achieve a cell concentration between 20-100 million cells per 1 mL.
• 18.
  Add DNA to cell solution at a concentration between 100-200 μg/mL total, keeping similar ratios of plasmids as discussed prior (step 1 for piggyBac and introduction for MADR).

  1. For the OC-100×2 cassette, a 50μl reaction will be prepared for each well. Using the concentrations above, a 50μl reaction would contain between 1-5 million cells and 5-10 μg total DNA.
• 19.
  Insert the cassette into the MaxCyte electroporator and select the appropriate program for the cell type.

  1. Electroporation programs will need to be empirically tested for specific cell lines and plasmids. Programs used to transfect iPSCs in order from very strong to mild energy are Opt 0-6, Opt 0-5, Opt 0-4, Opt 0-3. The very strong, high-energy programs will result in lower viability and may require a higher starting cell concentration. The milder protocol Opt 0-3 may need more DNA, up to 300 μg/mL. Consult with MaxCyte specialists to find the proper program for your application.

Optional: Recovery stage after in vitro transfection. (Performed after step 6 if Lonza nucleofector is used and step 19 if MaxCyte electroporator is used, then return to step 8)

• 20.To increase cell viability, the following optional recovery step may be performed after transfection.
• 21.
  Directly to the transfection cassette, add warm growth media (“recovery media”) to the surface without pipetting or disturbing the cells.

  • a
    Use caution adding additional media if your cassette contains multiple wells, such as with the MaxCyte OC-100x2 cassette. Care should be used to not overflow into neighboring wells.
• 22.
  If an excessive amount of DNA was used during transfection, DNase can also be added to the warm recovery media to digest exogenous DNA remaining after transfection to avoid DNA cytotoxic effects. 1 unit of DNase can degrade 1μg of DNA in 10 minutes at 37°C (check manufacturer’s instructions). Alternatively, DNase can be added at 5% the volume of the reaction.

  • a
    Additionally, add ROCK inhibitor at a working concentration of 10 μM to the recovery media if iPSCs are being transfected.
• 23.Carefully move the transfection cassette (with recovery media) into an incubator at 37°C for 20-30 minutes.
• 24.
  After rest, plate cells in the desired culture vessel as described above.

  1. Add additional ROCK inhibitor to maintain a working concentration of 10 μM to the culture media if iPSCs are being transfected.
• 25.Change media the day after transfection to remove debris and dead cells.

BASIC PROTOCOL 3

ADDING DOXYCYCLINE TO CELLS TO INDUCE/REVERSE GOI

The addition of doxycycline to the culture medium (or diet of animal) harboring cells with the Tet-GOI system causes the transactivator to undergo a conformational change and bind the TRE-Bi promoter. This facilitates transcription of the GOI and clover/luciferase reporters (when the rtTA-v10 Tet-On transactivators is used). When doxycycline is no longer present, the Tet-On transactivator no longer binds the TRE-Bi promoter, and transcription is no longer induced–hence the system is inducible and reversible. Furthermore, the system allows robust inducibility with minimal leakiness, allowing for the native fluorescence of the BFP, clover, and mScarlet reporters to be observed without immunocytochemical staining (Fig. 2).

Figure 2.

The Tet-GOI system allows for robust gene expression after the addition of doxycycline (dox) with minimal leakiness. (A-B) Human iPSC-derived NPCs nucleofected with the pB-tet-GOI system grown in the presence (A1-A3) or absence (B1-B3) of dox for 4 days (n.f. = native fluorescence, cells were fixed and imaged unstained). (C-D) Mouse NPCs nucleofected with the MADR-Tet system. (C2) Induced Tet-on reporter (c-Myc epitope tag) after 4 days of 200ng/mL doxycycline administration. (D2) Lack of inducible reporter when no doxycycline is administered. (C3 & D3) Transfected cells constitutively express mScarlet indicating successful transfection with Tet-GOI system.

For in vivo studies, either the piggyBac and MADR based Tet-GOI systems can be used to incorporated DNA constructs into cells in the postnatal CNS by several methods, including (1) transplanting nucleofected cells generated in the in vitro aspect of this protocol or (2) in vivo electroporation of the plasmids directly into cells lining the lateral ventricle (Rincon Fernandez Pacheco et al., 2020; Kim et al. 2019; Akhtar et al. 2015, 2018) or pial surface (Breunig et al. 2012; Levy et al. 2014). To induce/reverse GOI activity, mice can be fed with doxycycline chow/water or orally gavaged with a doxycycline solution. The oral gavage method ensures an accurate amount of doxycycline is received and allows experiments to be carried out within littermates sharing the same housing environment (i.e., with some littermates receiving doxycycline and others not receiving doxycycline). Mouse chow containing doxycycline (dox chow) can be used to induce GOI expression in many animals at once. Dox chow has the advantage of being less time consuming than performing oral gavages. The disadvantage of dox chow compared to oral gavage is the whole cage must be put on dox together, as well as having less control over the exact amount of dox administered (rodent chow containing varying amounts of doxycycline are manufactured) (Redelsperger et al., 2016).

NOTE: All animal handling should be carried out after seeking IACUC approval for the respective procedures. Training from personnel experienced with animal handling and proficient in the respective procedures is recommended.

Materials

Doxycycline (dox; Clontech, cat. no. 631311)

Doxycycline mouse chow - 200mg/kg (200ppm red) (Envigo, cat. no. TD.180625)

Cell culture growth medium for neural progenitor cells (from Basic Protocol 2; see recipe in Reagents and Solutions)

Phosphate-buffered saline (PBS; Sigma, cat. no. D8662)

CD1 Mice – used for piggyBac experiments (Charles River, Crl:CD1(ICR))

mTmG Mice – used for MADR experiments (Jackson Lab, strain no. 007576)

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

Protocol steps with step annotations

Adding dox to cell culture

1. Make stock doxycycline (dox) solution of 1 mg/ml (1 μg/μl) by dissolving dox powder in diH₂O. Store at 4°C in a dark tube (or clear tube wrapped in aluminum foil) to prevent light exposure. Fresh dox solutions should be made every 2 weeks.

2. Add dox to cell growth medium (From Basic Protocol 2, see recipe in Reagents and Solutions) at a final concentration of 100 ng dox/ml medium. (e.g., add 1 μl of 1 mg/ml dox solution to 10 ml medium). For inducible expression (using rtTA-V10 transactivator), the effect of dox (GFP and GOI expression) should be apparent within 24 hr. Reversible expression (using TTA2 transactivator) may require several days as clover protein may build up in the cell.

3. If continuous expression is desired, it is recommended to add dox to fresh medium during medium changes (every 2 to 3 days for human iPSC-derived NPCs).

4. To wash out dox, remove all medium and wash the cells twice every 24 hr, each time with 10 ml PBS. Depending on cell type and the half-life of the fluorescent protein, wash out time can be 4-14 days (Ayala-Sarmiento et al., 2020; Kim et al., 2019; Muzumdar et al., 2007).

5. Cells can be fixed and stained for desired proteins, or native fluorescence of BFP and clover can be imaged.

Administering dox to mice by oral gavage

• 2For oral gavage, make a 5 mg/ml (5 μg/μl) solution of dox in diH₂O. Store at 4°C in a dark (or aluminum foil covered) tube. Fresh dox solutions should be made every 2 weeks.
• 3Determine the mass (in grams) of mice to be gavaged. Suggested starting doses begin with a low dose of 15 μg dox/g mass whereas a high dose consists of 33 μg dox/g mass. Dosages must be empirically tested as the mouse species, transfected cell population, and DNA constructs all influence the transactivator response. The lowest dose which elicits a response should be use. For a 10 g mouse, a low dose would be 30 μl, and a high dose would be 66 μl, using a 5 mg/ml stock solution. The concentration of the stock solution can be increased if animal mass is above 20 g.
• 4Attach a blunt-tipped feeding tube to a 1-ml syringe. Re-useable or disposable feeding tubes are available. The disposable version is preferred for ease of use.
• 5Pre-wet the syringe and feeding tube with dox solution to avoid air bubbles.
• 6Using a blunt-tipped feeding tube attached to a 1-ml syringe, slightly tilt the head back and gently place the feeding tube in the mouth of the animal.
• 7The feeding tube should be inserted slightly lateral from midline. This will decrease the chances of the mouse biting the tube. The feeding tube should be angled slightly dorsal (toward the spine) to avoid the trachea. Little to no resistance should be encountered, as resistance may indicate that the tube is entering the trachea. Allow the feeding tube to fall into the esophagus by gravity. Do not apply pressure as this can cause perforation or scarring of the tissue.
• 8Once the feeding tube is properly inserted, dispense the desired amount of dox solution. Carefully remove the feeding tube and place the animal back in its cage.
• 9
  Mice should be gavaged every 2 to 3 days (2 to 3 times a week). Depending on the size of the animal, the number of times gavaged, and the concentration of solution, the washout period can range from 2 to 5 weeks, depending on if animals were given a low dose or high dose of dox.

  Initial transgene activation using low dose 15 μg dox/g oral gavage has been observed as early as one day post treatment in neonatal mice (Akhtar et al., 2018). The long-term ability of dox to cross the blood brain barrier in adult mice after neonatal electroporation has not been assessed by our group.

Administering dox to mice by dox chow

• 2
  Administering rodent chow containing doxycycline in place of oral gavage is preferable for large studies over long periods as it avoids multiple oral gavages a week.

  1. Pups should be weaned first before the administration of dox chow as breeding female mice should not be administered doxycycline.
• 3Store dox chow at 4°C, in accordance with IACUC and institutional animal housing standards, and use before expiration date.
• 4Exchange normal rodent chow for dox chow to start GOI inducible expression.
• 5
  Replace with fresh dox chow at minimum every week, or when low.

  1. Dox chow has not been assessed for short <48hr experiments as small but significant delays in feeding have been observed in some animals. In previous experiments, transgene expression appears to peak around 6d post-Dox administration (Akhtar et al., 2015).

BASIC PROTOCOL 4

ASSESSING GENE EXPRESSION IN VIVO BY NON-INVASIVE BIOLUMINESCENCE IMAGING OF LUCIFERASE ACTIVITY

The addition of luciferase linked to the clover transgene allows for non-invasive assessment of inducible gene expression. Administration of dox by oral gavage to mice that were electroporated on post-natal day 3 (P3) results in robust bioluminescence signal, whereas no detectable signal was observed in animals that did not receive the dox (Fig. 3A–C). Neural precursor cells targeted by electroporation of the lateral ventricle populate the striatum with glia and migrate to the olfactory bulb, where they differentiate into neurons (Fig. 3D) (Carleton, Petreanu, Lansford, Alvarez-Buylla, & Lledo, 2003). Assessment of the olfactory bulb of electroporated animals revealed native clover expression only in animals that received doxycycline, whereas nuclear BFP+ cells were observed in both dox and no dox groups. This suggests that the system is tight, non-leaky, and also corresponds to the luciferase expression assessed by bioluminescence analysis (Fig. 3E,F,G).

Figure 3.

Non-invasive in vivo bioluminescence imaging reveals luciferase activity after doxycycline (dox) administration and corresponding clover expression. (A, B) Total flux in animals assessed on postnatal day 18 (P18) after two doses of dox (15 μg dox/g) administered by oral gavage over 4 days on postnatal day 14 and 17 (P14 & P17). Hindlimb was assessed as background control. (C) Cartoon depicting in vivo piggyBac experimental timeline with a postnatal day 2 (P2) electroporation, postnatal day 14 and 17 (P14 & P17) oral gavage, and postnatal day 18 (P18) euthanasia and imaging. (D) Sagittal cartoon depicting the migration of neural progenitors along the Rostral Migratory Stream (RMS) to the olfactory bulb, where images of our region of interest are shown. Olfactory bulb dissected and imaged immediately after bioluminescence analysis. (E, F) Confocal imaging of olfactory bulbs of animals that were assessed for bioluminescence in panel Fig 3A. (E2) Displays induced Clover expression only in animals administered dox whereas there is no clover expression in animals not administered dox (F2). (n.f. = native fluorescence. Brains were fixed, sectioned at 70 μm and imaged unstained). (G) Electroporation using the MADR-Tet plasmid from Figure 1C2. Electroporation on postnatal day 2 (P2), dox chow administered postnatal day 28 (P28), and 7 days later on postnatal day 35 (P35) animals were euthanized and tissue was processed for staining. (G1) 20x stitched image of olfactory bulb after 7 days (7D) Dox, induced Myc reporter in green, NeuN staining in red. (G2-G4) 60x insert displaying neurons in the olfactory bulb expressing the inducible reporter Myc in green after 7D Dox.

Materials

VivoGlo Luciferin (Promega, cat. no. 1043)

10 mM HEPES, pH7.5 (Sigma, cat. no. H0887-100ML)

Mice that were transfected in Basic 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

IVIS Spectrum In Vivo Imaging System

Protocol steps with step annotations

1. Dilute 1 M HEPES solution to 10 mM and confirm a pH of 7.5. Reconstitute VivoGlo luciferin to a final concentration of 100 mM in 10 mM HEPES, pH 7.4. Transfer into 1-ml dark tubes and store up to 3 months at −20°C.

2. Determine the mass of mice to be imaged. Using clipper machine, shave the heads of the mice the day before (or day of) imaging.

3. Determine the volume of VivoGlo to be injected into each animal (150 mg/kg mass, e.g., 95 μl into a 20 g mouse). Before anesthetizing mice with isoflurane, pre-dispense the required amount into 1/2-ml syringe equipped with a 27-G needle. Use separate syringes for each animal.

4. Anesthetize the animals by placing into an isoflurane chamber. Once anesthetized, subcutaneously inject the required amount of VivoGlo in the neck/upper-back area. Record the time animals were injected. Safely dispose of syringe.

5. For the first imaging session, it is encouraged to determine the optimal incubation time of VivoGlo and the optimal exposure time. This can be done by incubating for 1 min and then obtaining continuous sequential images at “Auto Exposure” for 20 min. Determine the peak incubation and exposure time based on the luminosity intensity curve. Hindlimb was assessed as background control.

6. For increased accuracy and validity, it is encouraged to maintain the same incubation and exposure time for the duration of the study. Place additional mice for the study into the imager and image use the exposure time that was empirically determined in the previous step.

7. After imaging, return mice to their respective cages. Ensure mice are fully awake and behaving normally before returning cage to housing area.

REAGENTS AND SOLUTIONS

Use deionized, distilled water in all recipes.

Medium for human iPSC-derived neural progenitor cells

70:30 DMEM:F12 (350 ml DMEM and 150 ml F12)

DMEM (Fisher Scientific, cat. no. 11-965-092)

F12 Nutrient Mix (Fisher Scientific, cat. no. 11765070)

1% (5 ml) antibiotic-antimycotic (Life Technologies, cat. no. 15240-062)

5 μg/ml Heparin (Sigma, cat. no. H3149)

2% B-27 without Vit A (Life Technologies, cat. no. 12587-010)

100 ng/ml EGF (Sigma, cat. no. E9644)

100 ng/ml FGF (Millipore, cat. no. GF003)

Store up to 2 weeks at 4°C

COMMENTARY

Background Information

The response plasmid and transactivator plasmid of interest (rtTA-v10 for example) can be cloned into one large plasmid, as displayed with the pDonor MADR plasmid. This will eliminate the possibility of cells not being inducible due to receiving only the response or transactivator plasmids—as both are necessary for activation of the GOI and it is common for a percentage (<20%) of cells to receive only one plasmid when multiple plasmids are transfected (Loulier et al., 2014). Furthermore, it limits the possibility that cells containing only the response or transactivator plasmids may not proliferate as fast, as previous reports have suggested that other transactivator variants (not used in this study) are toxic to the cell (Morimoto & Kopan, 2009). While other studies have cautioned the use of dox at extremely high doses, we have not observed any abnormal affects at the dosages used in our study, although mitochondrial health was not assessed (Moullan et al., 2015).

The authors have validated both the piggyBac and MADR transgenic systems in human and mouse neural progenitor cells in vitro up to six weeks after nucleofection, and in vivo in the postnatal brain of neonatal mice up to six weeks after electroporation. However, long term analysis in vitro or in vivo has not been performed. Furthermore, the potential toxicity of the constitutive transactivator protein for extended periods of time has not been assessed.

While our study focused on the use of neuronal transcriptional regulators as the respective GOI, the system may be expanded to inducibly express elements to knockdown/delete genes such as shRNAs or CRISPR/Cas9. Furthermore, regulating trophic factor secretion may lend well to therapeutic models of neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS) and Parkinson’s Disease (PD), where increased secretion of growth factors have been reported to promote neuronal survival (Akhtar et al., 2018; Behrstock et al., 2006; Chtarto et al., 2016; Suzuki et al., 2007). Altogether, this technology provides an approach to regulate gene expression by addition of dox with relatively few side effects expected at the dosages given. Drawbacks of the tetracycline inducible system may include non-uniform expression of the GOI in a heterogenous population of cells as well as potential silencing of the transactivator promoter in certain cell types with certain plasmid constructs. Additionally, proper controls should be used to confirm the Tet response promoter does not have significant “leaky” expression in the absence of doxycycline (Akhtar et al. 2015). Finally, dox can have non-specific effects on cell populations and will likely alter the global microbiome (Boynton et al., 2017; Hill et al., 2021; Luger et al., 2018). Thus, controls such as a “No dox” condition or “dox inducible genetic reporter [without additional perturbation] with dox” are recommended to appropriately credential perturbations (Akhtar et al. 2015, 2018).

Adaptations to this protocol involve using the novel transgenic technology MADR, which has several advantages over the piggyBac system. The main purpose of MADR is to insert single copies of transgenes in a controlled fashion, in contrast to piggyBac which randomly integrates multiple copies throughout the genome. Controlling for a physiological normal level of gene expression is important to maintain the normal function of transgenic proteins. Overexpression of proteins has been shown to produce an imbalance of protein interactions, changing their function, and leading to a spectrum of disorders (Gibson et al., 2013). Additionally, when transfecting oncogenes, it is important to control for the copy number variant (CNV) as many disorders are related to the levels of protein expression (Collins et al., 2022). The multiple copy number insertions seen with piggyBac can be advantageous in certain circumstances when the overexpression of a gene is desired, such as with a fluorescent reporter when increased signal intensity is desired.

Another drawback of using a transgenic system which undergoes random site integrations, such as piggyBac, is that if an insertion occurs in the middle of a gene or promoter, an insertional mutagenesis (indel) can occur, which disrupts the function of a normal gene. This is controlled for using MADR since the donor plasmid can only integrate between LoxP and FRT sites, preventing indels from occurring. Additionally, MADR has a very large payload capacity, with pDonor plasmids tested up to ~20kbp and an upper limit that is only dictated by the biophysics of recombinant DNA technology and capabilities of the cognate recombinases to mediate insertion (e.g., BAC or artificial chromosome-delivered insertions should be feasible).

Critical Parameters

It is preferred that methods of plasmid delivery be used that have been verified for the specific cell type being used. Titrations of the DNA and cell concentrations many need to be performed to find optimal levels for each context. The system is compliant in methods of gene delivery that allow for the transfection of larger DNA fragments (10 kb). Researchers who intend to administer dox by oral gavage to mice should be proficient in the technique to ensure safety to the animal and researcher. It is recommended that appropriate training is acquired from animal care staff before attempting the protocol.

Troubleshooting

An important aspect of successful use of the pB-tet-GOI system is the accurate titration of the ratios of transactivator-to-response plasmids used for transgenesis and the amount of dox added. This will need to be empirically determined for each respective cell type. The authors have observed that using 1 μg of transactivator (instead of 3 μg) per nucleofection reaction resulted in minimal induction of transgenes upon addition of dox in human neural progenitor cells. For in vivo postnatal electroporation, a ratio of 1:1:0.5, response:transactivator:pBase plasmids has been tested and validated. Other ratios have not been tested. For MADR transgenesis, a range of ratios for pDonor:dual recombinase was tested and a ratio of 10:1 was found to have an advantageous balance of MADR-integrated cells versus total electroporated cells (Kim et al., 2019).

• Low viability after in vitro transfection: Large amounts of cell death can occur after in vitro transfection and each cell type and DNA mix needs optimization to increase viability. Tips:

  • Increase number of cells per transfection reaction.
  • Perform a transfection reaction without DNA. If the reaction without DNA has better viability, it means the DNA concentration maybe too high and is resulting in cytotoxicity. Try the optional step discussed using DNase after transfection.
  • Culture a small sample of cells taken prior to loading the transfection cassette. This pre-transfection population would reveal if one of the disassociation and handling steps is too harsh, resulting in low viability due to these initial steps and not the transfection.
• Low in vitro transfection efficiency: Cells are viable after transfection, but very few cells express the constitutive active reporter (TagBFP2 in this example). Tips:

  • Check reagents are fresh and compatibility of plasmids (especially in the case of MADR, which needs LoxP and FRT sites for integration). Note: even the orientation of the LoxP and FRT sites is critical. Using even a single recombinase site of the pair with the wrong orientation will prevent insertion.
  • Run the plasmids on an agarose gel to confirm proper plasmid length and perform gel-purification to remove supercoiled DNA.
  • Sequence whole plasmids, preferably with a service which performs QC and purity checks and compare with plasmid maps. Check for transgene or plasmid dimers.
  • Prepare DNA using an endotoxin-free prep kit.
  • Adjust microscope exposure as constitutive active fluorescent reporters have been observed to be dim in certain cells and DNA constructs.
  • Care should be taken when preparing plasmid mixes as a viscous DNA mix could affect the accuracy of plasmid ratios, resulting in a low transfection efficiency.
• Doxycycline administration results in a low gene expression: If the constitutively active reporter is observed, but the cells do not respond to doxycycline induction, this indicates something is preventing the activation of the Tet response promoter. Variable gene expression in vitro has been observed in different cell lines as well as with differing GOIs within the same cell line. The cause of this variability is still under investigation, although some avenues to troubleshoot are below:

  • Create doxycycline solutions fresh, protect from light, and avoid freeze-thaw cycles.
  • Empirically test titrations of doxycycline starting from 1 μg/mL to 100 ng/mL.
  • Adjust microscope exposure as inducible fluorescence reporters have been observed to be low in certain cells and with certain DNA constructs.
  • For in vitro studies, the selection of a different transfected clonal population may result in a cell population with a different response.
  • For in vivo postnatal electroporation, low gene expression may be the result of poor DNA mixture or improper electroporation technique. Plasmid mixes should be prepared fresh and contain the proper ratios of plasmids (ensure proper pipetting volume with DNA mixes that are viscous).
  • Silencing of the tet-on construct has been reported in certain circumstances.
• Potential silencing of the Tet promoter: Silencing of the inducible Tet promoter has been occasionally observed in certain cell types (Gödecke et al., 2017; Ordovás et al., 2015).

  • The use of Histone Deacetylase Inhibitors (HDAC) such as Trichostatin A, Valproic Acid, and Sodium Butyrate have been reported in the literature to attenuate inducible Tet expression based on epigenetic modifications (Mali et al., 2010). It was reported sub-millimolar concentrations of sodium butyrate increased inducible fluorescent reporter expression.

Understanding Results

The authors have successfully employed the above technique to inducibly express transcription factors, oncogenes, nucleases, short hairpin RNAs, and secreted factors as the GOI. MADR has been showcased in mammalian cells both in vitro and in vivo to deliver a variety of transgenes (Akhtar et al. 2015; 2018; Kim et al., 2019).

Protocol 1 discusses the cloning of a tetracycline response plasmid in which a successful cloning project will result in the growth of a bacterial colony on an agar plate with the appropriate antibiotic for selection. Lack of colonies after plating could indicate an incorrect ligation of fragments, which results in a plasmid lacking the antibiotic resistance gene used for selection. Plates should be incubated for an appropriate time as the selection antibiotic will deteriorate over time with heat. Additional care should be taken to avoid selecting “satellite colonies”, when a large bacterial colony degrades the antibiotic in the area and results in the growth of small nearby “satellite colonies”, which may lack the correct antibiotic resistance gene. Sequencing DNA extracted from the bacterial colonies should be used to confirm correct sequences devoid of mutations, especially in regions which underwent PCR.

Protocol 2 explains how to transfect neural progenitors with the plasmid designed in protocol 1. Successful transfection will result in a cell which fluoresces with the constitutively active fluorescent protein reporter. Some reporters may be visible after 24 hours, but typically fluorescence is checked after 3 days to allow for the accumulation of fluorescent proteins. Minor episomal expression of fluorescent proteins is possible, which will wash out over the next 1-2 weeks and only cells with proper integration will have persistent and stable fluorescent protein expression. Certain cell types, including iPSCs, have resulted in a very low expression of fluorescent proteins, so care should be taken when scanning the plates to ensure proper exposure time is set to observe the fluorescent protein. Difficulties with this protocol typically arise from low viability and/or low transfection efficiency and optimization and troubleshooting may need to be performed.

Protocol 3 addresses inducing expression of genetic elements of interest (GOI) using doxycycline. Successful induction should result in expression of the fluorescent protein reporters typically around day 3, but sometimes can be observed as soon as 24hr after doxycycline administration. Care should be taken when examining in vitro cultures on an epifluorescence scope to ensure laser strength and exposure time is appropriate to observe the reporters, but not strong enough to observe autofluorescence or apoptotic cells. True signal should be isolated to parts of the cell in which the fluorescent proteins are shuttled (i.e. membrane, nuclear, cytoplasm, mitochondria, lysosomal, etc.). Finally, care should be taken not to expose cells to UV light for long periods (e.g. to detect TagBFP2) as this will cause DNA damage and cellular toxicity.

Protocol 4 describes a technique to use luciferase to non-invasively examine gene expression in vivo in a mouse brain. Studies which result in a large population of cells, such as a tumor, will elucidate a strong signal detectible from the imaging system. If the population of induced cells is smaller, luminescence may be difficult to detect over background signal. Proper incubation time of VivoGlo Luciferin should be performed, and the same variables should be used throughout the experiment. Confirmation of gene expression can be performed by immunofluorescence staining of luciferase (Akhtar et al. 2018).

Time Considerations

Subcloning of transgenes into the Tet-GOI system and DNA purification can be accomplished within roughly two weeks, allowing for sequence verification and maxiprep of plasmid. Transduction of cells in vitro and selection of stable cells is typically done over the course of three weeks. Specifically, TagBFP2+ cells are FAC-sorted on day 7 and then 2 weeks later, permitting for the dilution of episomal pBase and unintegrated plasmids. Alternatively, antibiotic selection can be employed over roughly a similar timeframe. In vivo, transgene expression can be detected within hours of induction when administering doxycycline with oral gavage (Akhtar et al., 2018). Oral gavages should be performed every 3-4 days for the length of the experiment. Mouse chow containing doxycycline should be refreshed once a week and the earliest timeframe tested for induction has been 3 days. Induction kinetics and reversal after dox removal will depend greatly on dox dosage/frequency and the degradation rate of each individual transgenic protein.

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.