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Cre-Dependent Adeno-Associated Virus Preparation and Delivery for Labeling Neurons in the Mouse Brain Z. Josh Huang, Hiroki Taniguchi, Miao He and Sandra Kuhlman Cold Spring Harb Protoc; doi: 10.1101/pdb.prot080382 Email Alerting Service Subject Categories
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Protocol
Cre-Dependent Adeno-Associated Virus Preparation and Delivery for Labeling Neurons in the Mouse Brain Z. Josh Huang, Hiroki Taniguchi, Miao He, and Sandra Kuhlman
Virus-mediated gene delivery is a powerful strategy for labeling and manipulating neurons in mammalian brains. A major drawback of this gene delivery method has been the lack of cell-type specificity. However, methods that combine Cre-knockin mice and Cre-activated adeno-associated virus (AAV) have now been developed to achieve high-level, stable, and cell-type-specific gene expression. Here, we describe a protocol for the design, production, and delivery of Cre-dependent AAVs. This method is simple and highly efficient, and allows chronic live imaging of defined classes of synapses in the mouse brain.
MATERIALS It is essential that you consult the appropriate Material Safety Data Sheets and your institution’s Environmental Health and Safety Office for proper handling of equipment and hazardous material used in this protocol.
Reagents
Bleach for disinfectant (10%) Carprophen or other analgesic Column or cesium chloride gradient Ethanol (75%) Eye ointment Ketamine/xylazine Mice Cre, Flp driver lines Many driver lines are available from the Jackson Laboratory (http://www.jax.org/) or the Mutant Mouse Regional Resource Center (MMRRC) (http://www.mmrrc.org/). In addition, the National Institutes of Health (NIH) Neuroscience Blueprint supports several Cre driver projects (http://www.credrivermice.org/), and the Cre drivers will be distributed through the above two sources. The Gensat Project (http://www.gensat.org/index.html) is also generating a series of Cre drivers distributed through the MMRRC.
Reporter lines Most of the Cre- or Flp-dependent reporter lines are available from The Jackson Laboratory.
Tg GFP lines The Gensat Project has generated and has characterized hundreds of BAC Tg GFP lines (http://www.gensat.org/ index.html).
Saline (sterile) Adapted from Imaging in Developmental Biology (ed. Sharpe and Wong). CSHL Press, Cold Spring Harbor, NY, USA, 2011. © 2014 Cold Spring Harbor Laboratory Press Cite this protocol as Cold Spring Harb Protoc; doi:10.1101/pdb.prot080382
190
Downloaded from http://cshprotocols.cshlp.org/ at WASHINGTON UNIV SCHOFMED on June 21, 2014 - Published by Cold Spring Harbor Laboratory Press
Cre-Dependent AAV for Labeling Neurons in Mouse Brain
Tissue adhesive Virus (recommended concentration, >109 genome copies/mL) Recombinant adeno-associated virus (rAAV) can be produced at several commercial and academic laboratories such as Virapur (http://www.virapur.com/), the University of Pennsylvania (http://www.med.upenn.edu/gtp/ vector_core.shtml), and the University of North Carolina at Chapel Hill (http://genetherapy.unc.edu/services. htm). In addition, Agilent Technologies (http://www.genomics.agilent.com) provides a kit for producing AAV2/2. A detailed description of rAAV production and a protocol can be found in Grieger et al. (2006).
Equipment
Absorbent tissue or gel foam Biosafety Level 2 (BSL2) facility Cotton swabs Dental or microdrill and drill bits (carbide burr FG ¼) Equipment for chronic cranial window procedure including glass coverslips (5 mm; Electron Microscopy Sciences) and headbar (titanium or aluminum) (optional, see Step 10) Fiber-optic light source Forceps (#5; fine and coarse) Glass bead sterilizer Glass pipettes (pulled) Pipettes for injection should be pulled ahead of time. Pull pipettes so they have a long taper. Break the tip to 10 µm if using a Picospritzer or 20–50 µm if using the Nanoinjector system or another volume-displacement method.
Heating pad (feedback controlled) Insulin syringes Isoflurane vaporizer (optional, see Step 6) Micromanipulator Microruler Microscope We use a two-photon laser-scanning microscope with high-numerical aperture (high-NA; 0.8–0.95), 20×–60× water-immersion objectives. The light source is a tunable Ti:sapphire laser. Use the image acquisition software that is supplied with the laser-scanning microscope or software such as ScanImage. A dissecting scope is needed for the injections. For more information on the procedure for in vivo two-photon imaging of GFP-labeled neurons, see Holtmaat et al. (2009).
Pipette puller (e.g., Sutter P97) Pressure source for injection (e.g., Picospritzer or Nanoinjector syringe pump) Sutures (optional) Scalpel with blade #11 Scissors Stereotaxic frame
METHOD Viral Vector Design
1. Use either of the two strategies outlined below to render green fluorescent protein (GFP) expression conditional on Cre-mediated recombination. rAAV vector has a capacity of 5 kb for an exogenous DNA fragment. Conventional rAAV vectors often use a strong cytomegalovirus (CMV) or CAGG promoter to drive marker gene expression (e.g., GFP). AAVmediated expression is itself strong, so in some cases, a weaker promoter may be sufficient or more desirable.
i. Insert a loxP–STOP–loxP cassette between the promoter and the GFP gene (see Fig. 1A). There is some concern that a strong promoter might drive through the transcription STOP cassette, causing leaky expression in the absence of Cre, but results from multiple laboratories show that such leakiness of the STOP cassette seems quite minimal.
Cite this protocol as Cold Spring Harb Protoc; doi:10.1101/pdb.prot080382
191
Downloaded from http://cshprotocols.cshlp.org/ at WASHINGTON UNIV SCHOFMED on June 21, 2014 - Published by Cold Spring Harbor Laboratory Press
Z.J. Huang et al.
B PCMV
loxP STOP
GFP
A PCMV
GFP
PCMV
loxP
CRE
CRE
lox2272
PCMV GFP
GFP
FIGURE 1. Neuron labeling by GFP expression using Cre-activated AAV vectors. Two strategies have been used to render GFP expression conditional on Cre-mediated recombination. (A) A loxP-STOP-loxP cassette is inserted between the promoter and the GFP gene. (B) A GFP gene flanked by a pair of incompatible antiparallel loxP sites is cloned in the opposite orientation to an upstream cytomegalovirus (CMV) promoter; Cre recombination results in an inversion of this flip-excision (FLEX) switch and, thus, expression of the GFP gene.
ii. Clone a GFP gene flanked by a pair of incompatible antiparallel loxP sites in the opposite orientation to an upstream CMV promoter (see Fig. 1B). Cre recombination results in an inversion of this FLEX switch and, thus, expression of the GFP gene. This strategy eliminates the concern of Cre-independent leaky expression and also gives larger cloning capacity for marker genes.
AAV Production and Serotypes
2. Transfect a heterologous cell line (e.g., HEK293) with three plasmids: (i) the Tg plasmid (pLTR) containing the recombinant DNA insert, (ii) the adenovirus plasmid encoding replication/capsid DNA replication proteins, and (iii) the helper plasmid encoding adenovirus genes required for high-titer production in HEK293 cells. 3. Two to three days after transfection, harvest the virion particles, and purify them using either a column or a cesium chloride gradient. Measure the virus titer. A minimum titer of 1 × 109 genome copies/mL is necessary for in vivo mouse brain injections; a titer of 1011–1013 genome copies/mL is ideal. For most researchers, the two most important parameters of rAAV are serotype and titers. Detailed description of AAV serotypes and hybrid serotypes can be found in Grieger et al. (2006) and Choi et al. (2005). rAAV serotype 2 is most effective in transfecting neurons but, in its native form, has limited spread in tissues and is often restricted near the site of injection. Serotype 2 rAAV can be cross-packaged into different capsid serotypes (i.e., hybrid serotypes) to increase the area of transfection. Cross-packaging serotypes include at least 1–9 and rh10 (Cearley and Wolfe 2006; Cearley et al. 2008) and are denoted as AAV2/n. rAAV2/1 and rAAV2/9 have a much-increased spread compared with AAV2/2. Different capsids may also show varying degrees of tropism for different cells types.
AAV Delivery Most institutions require that AAV injections be performed in a BSL2 facility. Consult your institution regarding these isolation requirements and regulations regarding animal experimentation.
4. Load the pulled pipette with the virus just before anesthetizing the animal, and store it in a secure location. The pipette can be either front loaded (e.g., using the Nanoinjector system or via temporarily attaching tubing to the back end and a 10-mL syringe to generate negative pressure). Alternatively, the pipette can be back loaded using a Picospritzer. See Troubleshooting.
5. Sterilize the surgical instruments in a glass bead sterilizer (or autoclave). Sterilize the working surface with 75% ethanol, and remove any unnecessary objects from the surgery table. 6. Anesthetize the mouse with an intraperitoneal injection of ketamine/xylazine mixture (0.13 mg/g, 0.01 mg/g body weight) or, alternatively, by isoflurane inhalation (4%–5% induction, 1.5%–2% maintenance, oxygen flow rate of 0.5 L/min). 7. Trim the hair, and then place the animal on a heating pad. Mount the animal in the stereotaxic frame. Cover its eyes with eye ointment to prevent drying. 192
Cite this protocol as Cold Spring Harb Protoc; doi:10.1101/pdb.prot080382
Downloaded from http://cshprotocols.cshlp.org/ at WASHINGTON UNIV SCHOFMED on June 21, 2014 - Published by Cold Spring Harbor Laboratory Press
Cre-Dependent AAV for Labeling Neurons in Mouse Brain
8. Turn on the fiber-optic light source, being careful to minimize heat on the animal and on any tubing. Administer analgesics to the mouse at this time (e.g., carprophen subcutaneously, 0.007 mg/g body weight). 9. Disinfect the area in which the incision will be made using a cotton swab dipped in 75% ethanol. Also use the moist swab to remove any loose hair and repeat as needed. 10. Using scissors, make a small opening in the skin. If a chronic cranial window is to be used, cut away as much scalp as needed, prepare the bone surface, and perform a craniotomy as described in Holtmaat et al. (2009). 11. If a craniotomy is not being performed, make a small hole in the skull using a dental drill. Use a microruler to identify desired coordinates. 12. Lift away small bone fragments with a scalpel or fine forceps. Remove enough bone to clearly see the surface of the brain (
Cre-Dependent Adeno-Associated Virus Preparation and Delivery for Labeling Neurons in the Mouse Brain Z. Josh Huang, Hiroki Taniguchi, Miao He and Sandra Kuhlman Cold Spring Harb Protoc; doi: 10.1101/pdb.prot080382 Email Alerting Service Subject Categories
Receive free email alerts when new articles cite this article - click here. Browse articles on similar topics from Cold Spring Harbor Protocols. DNA Delivery/Gene Transfer (228 articles) DNA Delivery/Gene Transfer, general (261 articles) Imaging for Neuroscience (269 articles) In Vivo Imaging (274 articles) In Vivo Imaging, general (138 articles) Transgenic Mice (71 articles) Transgenic Mice, general (65 articles) Viral Methods (72 articles)
To subscribe to Cold Spring Harbor Protocols go to:
http://cshprotocols.cshlp.org/subscriptions
© 2014 Cold Spring Harbor Laboratory Press
Downloaded from http://cshprotocols.cshlp.org/ at WASHINGTON UNIV SCHOFMED on June 21, 2014 - Published by Cold Spring Harbor Laboratory Press
Protocol
Cre-Dependent Adeno-Associated Virus Preparation and Delivery for Labeling Neurons in the Mouse Brain Z. Josh Huang, Hiroki Taniguchi, Miao He, and Sandra Kuhlman
Virus-mediated gene delivery is a powerful strategy for labeling and manipulating neurons in mammalian brains. A major drawback of this gene delivery method has been the lack of cell-type specificity. However, methods that combine Cre-knockin mice and Cre-activated adeno-associated virus (AAV) have now been developed to achieve high-level, stable, and cell-type-specific gene expression. Here, we describe a protocol for the design, production, and delivery of Cre-dependent AAVs. This method is simple and highly efficient, and allows chronic live imaging of defined classes of synapses in the mouse brain.
MATERIALS It is essential that you consult the appropriate Material Safety Data Sheets and your institution’s Environmental Health and Safety Office for proper handling of equipment and hazardous material used in this protocol.
Reagents
Bleach for disinfectant (10%) Carprophen or other analgesic Column or cesium chloride gradient Ethanol (75%) Eye ointment Ketamine/xylazine Mice Cre, Flp driver lines Many driver lines are available from the Jackson Laboratory (http://www.jax.org/) or the Mutant Mouse Regional Resource Center (MMRRC) (http://www.mmrrc.org/). In addition, the National Institutes of Health (NIH) Neuroscience Blueprint supports several Cre driver projects (http://www.credrivermice.org/), and the Cre drivers will be distributed through the above two sources. The Gensat Project (http://www.gensat.org/index.html) is also generating a series of Cre drivers distributed through the MMRRC.
Reporter lines Most of the Cre- or Flp-dependent reporter lines are available from The Jackson Laboratory.
Tg GFP lines The Gensat Project has generated and has characterized hundreds of BAC Tg GFP lines (http://www.gensat.org/ index.html).
Saline (sterile) Adapted from Imaging in Developmental Biology (ed. Sharpe and Wong). CSHL Press, Cold Spring Harbor, NY, USA, 2011. © 2014 Cold Spring Harbor Laboratory Press Cite this protocol as Cold Spring Harb Protoc; doi:10.1101/pdb.prot080382
190
Downloaded from http://cshprotocols.cshlp.org/ at WASHINGTON UNIV SCHOFMED on June 21, 2014 - Published by Cold Spring Harbor Laboratory Press
Cre-Dependent AAV for Labeling Neurons in Mouse Brain
Tissue adhesive Virus (recommended concentration, >109 genome copies/mL) Recombinant adeno-associated virus (rAAV) can be produced at several commercial and academic laboratories such as Virapur (http://www.virapur.com/), the University of Pennsylvania (http://www.med.upenn.edu/gtp/ vector_core.shtml), and the University of North Carolina at Chapel Hill (http://genetherapy.unc.edu/services. htm). In addition, Agilent Technologies (http://www.genomics.agilent.com) provides a kit for producing AAV2/2. A detailed description of rAAV production and a protocol can be found in Grieger et al. (2006).
Equipment
Absorbent tissue or gel foam Biosafety Level 2 (BSL2) facility Cotton swabs Dental or microdrill and drill bits (carbide burr FG ¼) Equipment for chronic cranial window procedure including glass coverslips (5 mm; Electron Microscopy Sciences) and headbar (titanium or aluminum) (optional, see Step 10) Fiber-optic light source Forceps (#5; fine and coarse) Glass bead sterilizer Glass pipettes (pulled) Pipettes for injection should be pulled ahead of time. Pull pipettes so they have a long taper. Break the tip to 10 µm if using a Picospritzer or 20–50 µm if using the Nanoinjector system or another volume-displacement method.
Heating pad (feedback controlled) Insulin syringes Isoflurane vaporizer (optional, see Step 6) Micromanipulator Microruler Microscope We use a two-photon laser-scanning microscope with high-numerical aperture (high-NA; 0.8–0.95), 20×–60× water-immersion objectives. The light source is a tunable Ti:sapphire laser. Use the image acquisition software that is supplied with the laser-scanning microscope or software such as ScanImage. A dissecting scope is needed for the injections. For more information on the procedure for in vivo two-photon imaging of GFP-labeled neurons, see Holtmaat et al. (2009).
Pipette puller (e.g., Sutter P97) Pressure source for injection (e.g., Picospritzer or Nanoinjector syringe pump) Sutures (optional) Scalpel with blade #11 Scissors Stereotaxic frame
METHOD Viral Vector Design
1. Use either of the two strategies outlined below to render green fluorescent protein (GFP) expression conditional on Cre-mediated recombination. rAAV vector has a capacity of 5 kb for an exogenous DNA fragment. Conventional rAAV vectors often use a strong cytomegalovirus (CMV) or CAGG promoter to drive marker gene expression (e.g., GFP). AAVmediated expression is itself strong, so in some cases, a weaker promoter may be sufficient or more desirable.
i. Insert a loxP–STOP–loxP cassette between the promoter and the GFP gene (see Fig. 1A). There is some concern that a strong promoter might drive through the transcription STOP cassette, causing leaky expression in the absence of Cre, but results from multiple laboratories show that such leakiness of the STOP cassette seems quite minimal.
Cite this protocol as Cold Spring Harb Protoc; doi:10.1101/pdb.prot080382
191
Downloaded from http://cshprotocols.cshlp.org/ at WASHINGTON UNIV SCHOFMED on June 21, 2014 - Published by Cold Spring Harbor Laboratory Press
Z.J. Huang et al.
B PCMV
loxP STOP
GFP
A PCMV
GFP
PCMV
loxP
CRE
CRE
lox2272
PCMV GFP
GFP
FIGURE 1. Neuron labeling by GFP expression using Cre-activated AAV vectors. Two strategies have been used to render GFP expression conditional on Cre-mediated recombination. (A) A loxP-STOP-loxP cassette is inserted between the promoter and the GFP gene. (B) A GFP gene flanked by a pair of incompatible antiparallel loxP sites is cloned in the opposite orientation to an upstream cytomegalovirus (CMV) promoter; Cre recombination results in an inversion of this flip-excision (FLEX) switch and, thus, expression of the GFP gene.
ii. Clone a GFP gene flanked by a pair of incompatible antiparallel loxP sites in the opposite orientation to an upstream CMV promoter (see Fig. 1B). Cre recombination results in an inversion of this FLEX switch and, thus, expression of the GFP gene. This strategy eliminates the concern of Cre-independent leaky expression and also gives larger cloning capacity for marker genes.
AAV Production and Serotypes
2. Transfect a heterologous cell line (e.g., HEK293) with three plasmids: (i) the Tg plasmid (pLTR) containing the recombinant DNA insert, (ii) the adenovirus plasmid encoding replication/capsid DNA replication proteins, and (iii) the helper plasmid encoding adenovirus genes required for high-titer production in HEK293 cells. 3. Two to three days after transfection, harvest the virion particles, and purify them using either a column or a cesium chloride gradient. Measure the virus titer. A minimum titer of 1 × 109 genome copies/mL is necessary for in vivo mouse brain injections; a titer of 1011–1013 genome copies/mL is ideal. For most researchers, the two most important parameters of rAAV are serotype and titers. Detailed description of AAV serotypes and hybrid serotypes can be found in Grieger et al. (2006) and Choi et al. (2005). rAAV serotype 2 is most effective in transfecting neurons but, in its native form, has limited spread in tissues and is often restricted near the site of injection. Serotype 2 rAAV can be cross-packaged into different capsid serotypes (i.e., hybrid serotypes) to increase the area of transfection. Cross-packaging serotypes include at least 1–9 and rh10 (Cearley and Wolfe 2006; Cearley et al. 2008) and are denoted as AAV2/n. rAAV2/1 and rAAV2/9 have a much-increased spread compared with AAV2/2. Different capsids may also show varying degrees of tropism for different cells types.
AAV Delivery Most institutions require that AAV injections be performed in a BSL2 facility. Consult your institution regarding these isolation requirements and regulations regarding animal experimentation.
4. Load the pulled pipette with the virus just before anesthetizing the animal, and store it in a secure location. The pipette can be either front loaded (e.g., using the Nanoinjector system or via temporarily attaching tubing to the back end and a 10-mL syringe to generate negative pressure). Alternatively, the pipette can be back loaded using a Picospritzer. See Troubleshooting.
5. Sterilize the surgical instruments in a glass bead sterilizer (or autoclave). Sterilize the working surface with 75% ethanol, and remove any unnecessary objects from the surgery table. 6. Anesthetize the mouse with an intraperitoneal injection of ketamine/xylazine mixture (0.13 mg/g, 0.01 mg/g body weight) or, alternatively, by isoflurane inhalation (4%–5% induction, 1.5%–2% maintenance, oxygen flow rate of 0.5 L/min). 7. Trim the hair, and then place the animal on a heating pad. Mount the animal in the stereotaxic frame. Cover its eyes with eye ointment to prevent drying. 192
Cite this protocol as Cold Spring Harb Protoc; doi:10.1101/pdb.prot080382
Downloaded from http://cshprotocols.cshlp.org/ at WASHINGTON UNIV SCHOFMED on June 21, 2014 - Published by Cold Spring Harbor Laboratory Press
Cre-Dependent AAV for Labeling Neurons in Mouse Brain
8. Turn on the fiber-optic light source, being careful to minimize heat on the animal and on any tubing. Administer analgesics to the mouse at this time (e.g., carprophen subcutaneously, 0.007 mg/g body weight). 9. Disinfect the area in which the incision will be made using a cotton swab dipped in 75% ethanol. Also use the moist swab to remove any loose hair and repeat as needed. 10. Using scissors, make a small opening in the skin. If a chronic cranial window is to be used, cut away as much scalp as needed, prepare the bone surface, and perform a craniotomy as described in Holtmaat et al. (2009). 11. If a craniotomy is not being performed, make a small hole in the skull using a dental drill. Use a microruler to identify desired coordinates. 12. Lift away small bone fragments with a scalpel or fine forceps. Remove enough bone to clearly see the surface of the brain (
