Name: designing, packaging, and delivery of high titer crispr retro and lentiviruses via stereotaxic injection
Uploaded: 2016-05-23T13:00:00.000+00:00
Duration: 11 min 28 s
Description: Watch this Scientific Journal Video about Designing, Packaging, and Delivery of High Titer CRISPR Retro and Lentiviruses via Stereotaxic Injection at JoVE.com

This work was supported by the NINH grant R01MH097949 and the Autism Speaks Pilot Grant 7359 to BWL and the Norris Cotton Cancer Center Optical Imaging Shared Instrumentation Grant P30CA023108.

Ethics Statement: All protocols were approved by the Dartmouth Institutional Biosafety and Institutional Animal Care and Use Committee review boards.
1. Protocol for Designing a Guide Strand (sgRNA) for CRISPR/Cas9 Retrovirus
NOTE: There are many non-profit websites that can be used to generate sgRNAs to target a gene of interest (https://benchling.com/ and http://crispr.mit.edu/).&#160; The goal of this protocol is to design and order single stranded oligos from a commercial vendor that are annealed to each other. This annealed oligo will be ligated into the PXL transfer vector.&#160;See supplemental video 1 for an example of designing sgRNAs using Benchling.
<ol>
	<li>Use a website dedicated to designing sgRNAs. 
	NOTE: For example, inputting a coding/exonic sequence from near the start of the gene of interest into the website will generate a 20 nucleotide sgRNA. Use the 20 nucleotide sgRNA sequence to design the sense and anti-sense oligos that will be ordered to create the sgRNA.</li>
	<li>After generating the sgRNA, copy this sequence into a word processor. Add a G (guanine) to the beginning of the 20 nucleotide sgRNA sequence in the document if it does not already begin with one (i.e., G-20 nucleotide sgRNA sequence). This is necessary to ensure good transcription off the U6 promoter.</li>
	<li>Document the reverse complement of this now 20-21 nucleotide sequence. For the sense oligo, add &#34;CACC&#34; to the 5&#39; end of the sequence in the document (i.e., CACC-G-20 nucleotide sgRNA sequence). This overhang will be used to ligate the sequence into the PXL vector.</li>
	<li>For the antisense oligo, add AAAC to the 5&#39; end. Use this overhang to ligate the sequence into the PXL vector.</li>
	<li>Obtain the sense and antisense oligos. After receiving the oligos, make 100 &#956;M stocks using DNAse free water. Mix together 10 &#181;l each of the 100 &#956;M sense and antisense oligos with 4 &#181;l of 10x NEB Buffer 2, and 16 &#181;l of water. 
	NOTE: They do not need page purification or 5&#39;phosphorylation (as the BbsI overhangs are non-compatible cohesive ends).
	<ol>
		<li>Bring 200 ml of water to a boil in a 500 ml beaker, then float the tube containing this mixture in a foam &#34;floater&#34; holder. Allow the water to slowly cool from 95 &#176;C for 2 hr at room temperature.</li>
		<li>Dilute the now annealed-oligo mix 1:1,000 in sterile water and use immediately in the following ligation or store the remaining reaction at -20 &#176;C.</li>
	</ol>
	</li>
	<li>Digest pXL, a PX330 vector derivative, with the BbsI restriction enzyme at 37 &#176;C for 2 hr. Use a 40 &#181;l reaction containing 2 &#181;g of pXL, 4 &#181;l of 10x NEB Buffer 2, 1 &#181;l of Bbs1 and balance water. Subject the digested-pXL to routine gel purification using a commercial kit. Ensure that the yield is ~8.5 kB product.</li>
	<li>Using a commercial ligation kit, ligate 1 &#181;l of the 1:1,000 annealed-oligos with 50 ng of digested and gel purified-pXL. Transform the ligation product into competent recombination deficient E. coli (NEB 5-alpha, subcloning efficiency). Screen the transformants for the presence of the correct guide by plasmid DNA sequencing.</li>
	<li>Digest the U6, guide strand, and RNA scaffold elements (sgRNA) out of pXL using BstB1 and Pac1 restriction enzymes and ligate into the fluorescent protein-T2A-Cas9 viral backbone digested with the same restriction enzymes. Transform the ligation product (NEB 5-alpha maximum efficiency). The fluorescent protein-T2A-Cas9 viral backbone plasmids are low copy plasmids and thus low copy protocols must be used. 
	NOTE: A second sgRNA can be similarly introduced by PacI digestion of another pXL guide strand plasmid and ligated into the Pac1 digested and calf-intestinal phosphatase treated viral backbone already containing the first guide strand. Phosphatase treatment of the viral transfer plasmid will help reduce the number of transformants from the self-ligation of plasmids not containing the second guide.</li>
	<li>Sequence verify the final plasmid&#160;and maxi prep using the Nucleobond Xtra maxi prep kit and package it into a virus using the following &#34;Protocol for Retro/Lentiviral Production&#160;&#8212; CaPO4 Method&#34;.</li>
</ol>
2. Prepare 293FT/293GP Cells for Transfection (Retro/Lentiviral Production &#8212; CaPO4 Method)
<ol>
	<li>(Day 1) Rapidly thaw 1 vial of cells per 10 cm cell culture plate in a 37 &#176;C water bath. For lentiviral packaging, use 293FT cells. For retroviral packaging, use 293GP (gag/pol) cells.</li>
	<li>Pipette all of the thawed-cells from the cryo tube into a 15 ml conical tube and add 2 ml of pre-warmed CO2 equilibrated-complete Iscove&#39;s Modified Dulbecco&#39;s Medium.</li>
	<li>Centrifuge cells for 5 min at 500 x g to pellet. Aspirate the supernatant and resuspend the cell pellet in 10 ml of complete Iscove&#39;s Modified Dulbecco&#39;s Medium. Plate the cells onto a 10 cm cell culture dish. Incubate the cells overnight at 37 &#176;C in a 5% carbon dioxide incubator.</li>
	<li>(Day 2) 24 hr after plating, change media on the plate by aspirating the existing media and adding 10 ml of pre-warmed Iscove&#39;s Modified Dulbecco&#39;s Medium to the plate.
	<ol>
		<li>(Day 3-4) 24-48 hr after the media change and once the cells become confluent, split the cells to a confluency of 2.5-3.0 x 106 cells/plate (10 cm plate).</li>
		<li>To split the cells, aspirate the media and wash the plate with 5 ml of PBS. Add 1 ml of 0.25% trypsin to the plate and incubate at 37 &#176;C until the cells lift off the plate. Add 0.5 ml of Iscove&#39;s Modified Dulbecco&#39;s Medium to neutralize the 0.25% trypsin reaction and pipette the cells into a 1.5 ml tube.</li>
		<li>Spin the cells at 500 x g for 5 min. Resuspend the cells in 1 ml of Iscove&#39;s Modified Dulbecco&#39;s Medium. Dilute 10 &#181;l of cells in 90 &#181;l of PBS. Count the cells using either a hemocytometer or automated cell counter. Re-plate 2.5-3.0 x 106 cells/plate with complete Iscove&#39;s Modified Dulbecco&#39;s Medium. 
		NOTE: Cells will be ~50% confluent 24-34 hr after plating. Transfect the cells when they are ~50% confluent.</li>
	</ol>
	</li>
</ol>
3. (Day 5) CaPO4 Transfection and Viral Particle Collection
<ol>
	<li>Change the media 2 hr prior to transfection by aspirating the existing media and adding 10 ml of pre-warmed Iscove&#39;s Modified Dulbecco&#39;s Medium. Ensure that there is exactly 10 ml of media in the 10 cm plate.</li>
	<li>Prepare the transfection reagents for 2 dishes using 2, 5 ml round bottom polystyrene tubes. Label the first tube &#34;DNA&#34; and the second tube &#34;2X HBS&#34;. Adjust the concentration of DNA to 1&#956;g/&#956;l in Tris-EDTA at pH 7.4.
	<ol>
		<li>For lentiviruses, slowly add 20 &#956;l of transfer vector (the viral construct of interest), 13 &#956;l of CMVdelta8.9, 9 &#956;l of VSV-g , 860 &#956;l of molecular biology grade H2O, and 100 &#956;l of 2.5 M CaCl2 to the first tube (&#34;DNA&#34;) while continuously tapping on the tube to mix.</li>
		<li>For retroviruses, omit the CMVdelta8.9 plasmid. Add 20 &#181;l Transfer Vector, 15 &#956;l VSV-g, 860 &#956;l Molecular Biology Grade H2O, and 100 &#956;l 2.5 M CaCl2 to the tube labeled &#34;DNA&#34;.</li>
		<li>Add 1 ml of 2x HEPES-buffered saline (pH 7.0; this pH is absolutely critical) to the tube labeled &#34;2X HBS&#34;.</li>
		<li>Slowly add the 1 ml contents of the &#34;DNA&#34; tube to the &#34;2X HBS&#34; tube, one drop at a time. Continuously tap the &#34;2X HBS&#34; tube with the index or middle finger while adding the contents of the &#34;DNA&#34; tube. Observe apparent CaPO4 vesicles after each drop. Incubate the tube in the dark for 30 min at room temperature.</li>
	</ol>
	</li>
	<li>Add 1 ml of the transfection in slow droplets to each 10 cm cell plate, then incubate overnight at 37 &#176;C.</li>
	<li>(Day 6) Replace media with 8 ml of Iscove&#39;s Modified Dulbecco&#39;s Medium + 0.5% FBS and 40 mg Caffeine/100 ml media. If the transfer vector contains a fluorescent marker, then fluorophore expression indicates a successful transfection.</li>
	<li>(Day 7) Collect the media containing the viral particles by using a 10 ml serological pipette and dispense into a 50 ml conical tube. Store the 50 ml conical tube at 4 &#176;C. Add 8 ml of 0.5% FBS media to each plate.
	<ol>
		<li>(Day 8) Again, collect the media containing the viral particles and combine with the previous day&#39;s harvest in the 50 ml conical tube.</li>
	</ol>
	</li>
</ol>
4. Concentration and Purification of the Virus
<ol>
	<li>Make a 5x polyethylene glycol 6000 solution by adding 40% polyethylene glycol 6000 and 1.5 M NaCl to ddH2O. Autoclave the solution on the liquid cycle for 45 min at 121 &#176;C. Slowly mix the solution when cooling. 
	NOTE: The solution will start cloudy and then become clear as it cools.</li>
	<li>Centrifuge the 50 ml conical tube containing both collections of viral supernatant at 2,000 x g for 10 min in order to pellet insoluble material. Purify the viral media by filtering through a 0.45 &#181;m low protein binding syringe filter (PES or PVDF).</li>
	<li>Add 5x polyethylene glycol 6000 solution to media (The final concentration should be 8% polyethylene glycol 6000 and 0.3 M NaCl). Mix by inverting the tube several times (do not vortex). Incubate the viral containing polyethylene glycol solution at 4 &#176;C for 12 or more hr, remixing occasionally.</li>
	<li>(Day 9) Centrifuge the viral containing polyethylene glycol solution at 2,500 x g for 45 min. Remove and discard the supernatant and spin again for 2 min. Again, remove and discard the supernatant.</li>
	<li>Resuspend the pellet by adding 320 &#181;l of sterile phosphate-buffered saline (the 320 &#181;l is 1/100th of the original volume of viral containing media collected) and incubate overnight at 4 &#176;C. Optionally, resuspend the pellet at room temperature on a rocker for 30 min.</li>
	<li>After re-suspending the pellet, aliquot the virus (5-10 &#181;l per 0.5 ml tube) and freeze aliquots at -80 &#176;C (freeze thawing or storage at 4 &#176;C will dramatically reduce the titer).</li>
	<li>Titer the viruses using standard protocols, i.e., perform a dilution series on a 6-well plate of HEK293T cells and manually count fluorescent colonies 48 hr later.</li>
</ol>
5. Testing Efficacy of the CRISPR Virus
NOTE: Sequence clones using the following steps to test for production of double-strand breaks repaired by NHEJ using the mouse Neuro2A cells. This has the advantage over surveyor assays in that it can be used to determine the percentage of cells that have been modified and the nature of the indels resulting from NHEJ.
<ol>
	<li>Coat a 3.5 cm cell culture dish with a gelatinous protein mixture such as matrigel diluted 1:50 in Iscove&#39;s Modified Dulbecco&#39;s Medium and incubate for 30 min at 37 &#176;C. Aspirate the gelatinous protein mixture and plate the Neuro2A cells.</li>
	<li>After the Neuro2A cells reach 50% confluence, add the CRISPR Lentivirus or Retrovirus at a multiplicity of infection of 10 (i.e., 10 viral particles per cell). 
	NOTE: Neuro2A cells are not as amiable to infection as are HEK293 cells, thus, a single infection will not result in 100% of the cells being infected with the lentivirus. Further, the retrovirus only infects dividing cells and therefore will also not result in 100% infection. In order to achieve a near 100% rate of infection, addition of virus can be repeated on multiple days, splitting the cells as needed. Alternatively, fluorescent positive cells can be isolated using fluorescence-activated cell-sorting. The most effective way to achieve a 100% infection rate with a lentiviruses is to perform a single infection followed by FACS isolation. For a retrovirus, perform 4-rounds of infection 24 hr apart and follow by FACS isolation to ensure 100% infection.</li>
	<li>After infecting the cells for 1 week, expand the cells to near confluence on a 10 cm plate, aspirate the media, and wash the plate with 5 ml of phosphate-buffered saline. Add 1 ml of 0.25% trypsin and incubate at 37 &#176;C until the cells lift off the plate. Add 0.5 ml of Iscove&#39;s Modified Dulbecco&#39;s Medium to neutralize the 0.25% trypsin reaction and pipette the cells into a 1.5 ml tube and pellet the cells at 500 x g for 5 min.</li>
	<li>To isolate DNA, add 100 &#181;l of 50 mM potassium hydroxide, re-suspend the cells, and incubate at 95 &#176;C for 5 min. Neutralize the solution using 10 &#181;l of 1 M Tris, pH = 8.0.</li>
	<li>PCR amplify the region flanking the genomic region targeted by the CRISPR sgRNA using ~300 ng of the DNA isolated in step 5.5 using a high fidelity PCR master mix4.</li>
	<li>Run the PCR reaction on a 2.5% agarose gel and gel purify the appropriately sized fragment using a gel purification kit such as a gel DNA recovery kit. Ligate into a PCR cloning vector such as pGem-t-easy and transform into competent cells9.</li>
	<li>24 hr later, add 2 ml of LB broth into a 15 ml round-bottomed tubes as well as the appropriate antibiotic (ampicillin, neomycin, etc.). Inoculate the tube with an individual colony by using a sterile pipette tip or loop to select a single colony from the LB-plate. Expand and grow the colony by shaking the tube at 250 RPM and 37 &#176;C for 24 hr. Repeat for as many colonies as desired.
	<ol>
		<li>Using a mini-prep kit, isolate DNA from the E. coli and sequence with primers designed to amplify the area of the gene targeted by the sgRNA in order to analyze the Cas9 targeted region of the mouse genome.</li>
	</ol>
	</li>
</ol>
6. Stereotaxic Injection Protocol for the Adult Mouse
<ol>
	<li>Prepare for Surgery 
	NOTE: It is important to maintain sterile conditions during survival surgeries. This is accomplished in this protocol by heat sterilizing the surgery tools, using betadine to sterilize the injection site, and adding antibiotic ointment to the incision site after it is closed. It is also important to use sterile gloves as well as a thoroughly sterilized, dedicated surgery area.
	<ol>
		<li>Heat sterilize surgery tools prior to use by either autoclaving or a using hot bead sterilizer. Prepare recovery chamber and surgery area by turning on heating pads to maintain body temperature during surgery and recovery. Assemble the drill used to create holes in the skull for injection.</li>
		<li>Load 4 &#181;l of virus into injection needle by withdrawing the virus directly from the sterile PCR tube. Briefly remove the viral aliquot from ice. Maintain the virus in the syringe at room temperature for no more than 1 hr prior to injection.</li>
	</ol>
	</li>
</ol>
7. Stereotaxic Injection
<ol>
	<li>Confirm that the ventilation to the surgery suite is open to ensure proper air flow. Prepare the mouse for surgery by anesthetizing with 4% isoflurane in an induction chamber. Following anesthesia, shave the head to prepare the injection site.
	<ol>
		<li>Place the mouse in the stereotaxic instrument by using tweezers to move tongue down and to the side. Insert the bite bar into mouth until teeth drop into the slot, then secure the ear bars. Ensure that the body of the mouse is on the heating pad and the nose is situated in the nose cone. Direct 4% isoflurane into the nose cone. Confirm anesthesia by pinching the foot with tweezers and ensuring that there is no startle reflex.</li>
		<li>Apply artificial tears to lubricate the eyes. Finish preparing the injection site by swabbing the shaved head with alternating rounds of povidone-iodine and lidocaine.</li>
	</ol>
	</li>
	<li>Using a scalpel, cut small incision along center of scalp. Dry the skull with a swab and hydrogen peroxide if necessary to help visualize bregma.
	<ol>
		<li>Using a dissecting scope, locate bregma on the skull and place the drill tip on bregma. Zero (or record) the digital stereotactic coordinates on the x, y, and z planes.</li>
		<li>In order to ensure that the head is level on the rostral to caudal y-axis, place the drill bit on lambda and level the head so that the z-coordinate is roughly equal at both bregma and lambda.</li>
		<li>In order to ensure that the head is level on the x-axis, place the drill bit to y = 1/2 lambda. Ensure that the z-coordinate is equal at 1 mm on each side (x = +/-1 mm) of the sagittal suture. Adjust the skull if there is a difference in the z-coordinate at 1mm to the left and right of lambda.</li>
	</ol>
	</li>
	<li>Place the drill over the desired coordinates. For the dentate gyrus, use the coordinates y = -1.9 from bregma and x = +/- 1.1. Before drilling, lower the isoflurane to 2%, then slowly and carefully, drill through the skull.
	<ol>
		<li>After drilling all the holes, affix the filled-syringe onto the stereotaxic instrument. Visually center the syringe over the hole and zero the z coordinate at the skull.</li>
		<li>Slowly lower the syringe to deepest z-depth. For the dentate gyrus, the z depths are -2.5, -2.4, and -2.3. Begin injection at a rate of 0.25 &#181;l/min using a stereotaxic injector.
		<ol>
			<li>After the injection is complete at lowest Z-depth, wait 1 min, then raise to the next coordinate and begin injection again. Continue this pattern until all z-injection coordinates are injected. Wait 2 min before removing the syringe after the last injection. 
			NOTE: No adverse effects have been noted on the tissue by injecting up to 2 &#181;l of virus per hemisphere in the mouse brain.</li>
		</ol>
		</li>
		<li>Repeat injection for other bore holes.</li>
	</ol>
	</li>
	<li>After injection, remove the mouse from the stereotaxic instrument and suture the scalp with 6-0 silk sutures. Apply Lidocaine and an anti-bacterial cream to the wound.
	<ol>
		<li>Inject 0.8-1.0 ml saline + Ketoprofen (3-5 mg/kg) via I.P. route to manage pain. Then place mouse into heated recovery chamber. Do not leave the animal unattended until it has regained sufficient consciousness to maintain sternal recumbency. Do not return the animal to the company of other animals until it has fully recovered.</li>
	</ol>
	</li>
	<li>In the days following the surgery, weigh the mouse daily and provide soft food and treats. Check wound and note the general condition/demeanor.</li>
	<li>Following stereotaxic injection, the mice can be euthanized for slice prep electrophysiology1 or perfused and their brains removed, sliced, and stained via immunohistochemistry for analysis10.</li>
</ol>

<ol>
	<li>Williams, M. R., DeSpenza, T., Li, M., Gulledge, A. T., Luikart, B. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hyperactivity+of+newborn+Pten+knock-out+neurons+results+from+increased+excitatory+synaptic+drive.">Hyperactivity of newborn Pten knock-out neurons results from increased excitatory synaptic drive.</a> J Neurosci. 35 (3), 943-959 (2015).</li><li>Luikart, B. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pten+knockdown+in+vivo+increases+excitatory+drive+onto+dentate+granule+cells.">Pten knockdown in vivo increases excitatory drive onto dentate granule cells.</a> J Neurosci. 31 (11), 4345-4354 (2011).</li><li>Cong, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiplex+genome+engineering+using+CRISPR/Cas+systems.">Multiplex genome engineering using CRISPR/Cas systems.</a> Science. 339 (6121), 819-823 (2013).</li><li>Ran, F. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome+engineering+using+the+CRISPR-Cas9+system.">Genome engineering using the CRISPR-Cas9 system.</a> Nat Protoc. 8 (11), 2281-2308 (2013).</li><li>Sander, J. D., Joung, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CRISPR-Cas+systems+for+editing,+regulating+and+targeting+genomes.">CRISPR-Cas systems for editing, regulating and targeting genomes.</a> Nat Biotechnol. 32 (4), 347-355 (2014).</li><li>Luikart, B. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=miR-132+mediates+the+integration+of+newborn+neurons+into+the+adult+dentate+gyrus.">miR-132 mediates the integration of newborn neurons into the adult dentate gyrus.</a> PLoS One. 6 (5), e19077 (2011).</li><li>Nasri, M., Karimi, A., Allahbakhshian Farsani, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production,+purification+and+titration+of+a+lentivirus-based+vector+for+gene+delivery+purposes.">Production, purification and titration of a lentivirus-based vector for gene delivery purposes.</a> Cytotechnology. 66 (6), 1031-1038 (2014).</li><li>Ellis, B. L., Potts, P. R., Porteus, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Creating+higher+titer+lentivirus+with+caffeine.">Creating higher titer lentivirus with caffeine.</a> Hum Gene Ther. 22 (1), 93-100 (2011).</li><li>Fricano, C. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fatty+acids+increase+neuronal+hypertrophy+of+Pten+knockdown+neurons.">Fatty acids increase neuronal hypertrophy of Pten knockdown neurons.</a> Front Mol Neurosci. 7, 30 (2014).</li><li>Park, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lentiviral+vectors:+are+they+the+future+of+animal+transgenesis?.">Lentiviral vectors: are they the future of animal transgenesis?.</a> Physiol Genomics. 31 (2), 159-173 (2007).</li></ol>

The authors have nothing to disclose.

There are a few critical steps that are important for successful viral packaging. Cell health is critical before and during the transfection, as unhealthy cells will greatly reduce the amount of virus produced. If the transfection and packaging are successful, then 100% of the cells should express the fluorophore and the cells should form a functional syncytium. In step 3.2.4, tapping the tube is necessary for high-titer transfection efficiently, and the pH of the HEPES-buffered saline must be exact. The maxi-preps that produce the plasmids necessary for viral packaging must be extremely pure. To this point, it is helpful to ethanol precipitate the final DNA elution and re-suspend in Tris-EDTA buffer. It is also very important to reduce the amount of serum to 2% or less in the media that the caffeine is added to on Day 6 (step 3.4) before viral collection. If the serum is not reduced, then the final purified virus will contain an undesirable amount of serum protein. The use of polyethylene glycol 6000 when precipitating the viral particles excludes the necessity of ultracentrifugation. It is also important to note that the Cas9 CRISPR containing viruses typically have a titer around 10 fold less than viruses solely containing a fluorophore.
For the stereotaxic surgery, the use of inhaled anesthesia allows rapid and precise control or the animal&#39;s consciousness compared to injectable anesthetics and allows anesthesia over a larger age range. It is highly important to keep the surgical instruments clean and sterile, and reproducible targeting requires precise positioning of the head. Make sure there is no pitching or rolling of the head in the stereotaxic instrument and that the skull feels firmly in place. It can be useful to allow the skull to dry in order to find the sutures to determine the stereotaxic coordinates. Also, the rate and volume for each stereotaxic coordinate should be empirically determined.
This technique is limiting in that the spread of a lenti- or retrovirus is restricted, especially when compared to adeno-associated viruses (AAVs).Therefore, these viruses are valuable when infecting a discrete brain region, but not for overall infection associated with AAVs used for behavioral analysis in animals. The use of caffeine in this protocol greatly increases the titer of these viruses, but they are still not as high as the titers achieved in AAV packaging. Also, stable integration is only an advantage of fluorophore expression, as CRISPR/Cas9 forms stable genomic edits even when transiently transfected and it is possible that on-going expression of the Cas9 and sgRNA may eventually produce off target effects. The transient expression the CRISPR/Cas9 system with AAVs is sufficient to produce genomic changes that are propagated throughout cell divisions, however, fluorophore expression will not be maintained.
Creation of lenti- and retroviruses utilizing the CRISPR/Cas9 system will impart the ability to target any novel gene in a wide variety of organisms. The efficiency of gene editing appears to be dependent on the sequence of the guide RNA targeting the Cas9 cleavage. It has been empirically determined that between 10% and 80% of clones contain indels after sequencing infected Neuro2A cells. It is presently unknown whether indel frequencies calculated in Neuro2A cells are reflective of those in neurons. Guide RNA design software such as Benchling now include an &#34;on-target&#34; score that may be able to predict the efficiency of a given target sequence. To what degree such &#34;on-target&#34; scores are reliable needs to be empirically determined in neurons and other cell-types as the CRISPR-Cas9 system becomes more widely implemented.
Lentivirus-based transgenic animal production has been variably successful with reports that the lentivirus-delivered transgenes become silenced11. CRISPR mediated gene editing of DNA may be passed through the germ line to generate whole animal models. Thus, stable genomic editing may be achievable despite the silencing of viral-delivered fluorophores and Cas9 transgenes. This may provide an efficient platform for targeted genomic alterations. The viral delivery of the CRISPR/Cas9 system, while not requiring transgenic organisms, is complementary to those techniques. For example, injecting such viral particles into a compound transgenic animal that inducibly expresses Cre and Cre dependent opto- or chemo-genetic transgenes should facilitate complex studies into the relationship between genetic manipulations and neuronal activity. A second example is to deliver these Cas9/sgRNA viral particles into a conditional knockout in an attempt to screen for gene-gene interactions. Finally, another exciting route of this research is the screening of phenotypes and therapeutic compounds in patient derived cells, which can be used to validate and discover genetic networks that are disrupted in various diseases.

To study the basis of normal physiology and disease pathology, there is a need to precisely manipulate gene expression in model organisms. For mammalian model organisms, this is largely centered on the creation and development of transgenic mice wherein a genetic element of interest is flanked by sites recognized by a recombinase. This can result in a site specific manipulation of these flanked genes. While this has been a successful strategy, it is time and resource intensive; for example, creating a triple transgenic animal that would express a floxed gene, Cre recombinase, and a Cre reporter gene requires multiple matings and validation. In contrast, the stereotaxic injection of replication defective viral particles encoding a fluorescent protein and the recombinase into a floxed gene animal does not require complex genotyping or breeding strategies1. Further, if a fluorescent protein and Cre expressing virus is co-injected with a second virus encoding a different fluorescent protein, then this provides a within-tissue control for the targeted genetic manipulation. While this strategy still requires the use of knock-in animals, virally mediated RNA based strategies circumvent the need for transgenic animals. For example, stereotaxic injection of replication-deficient viruses that encode a fluorescent protein and a short hairpin RNA (shRNA) can use the cell&#39;s endogenous RNAi machinery to result in a potent reduction of the transcript of a gene of interest. However, shRNA strategies produce subtle gene knock-downs often resulting in modest cellular phenotypes2. While a knock-down may be more physiologically relevant for heterozygous gene dysfunction, its decreased robustness compared to a knock-out is not ideal for phenotypic discovery of novel genes.
A third technique that has recently emerged, the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeat)/Cas9 (CRISPR-associated protein 9) system, relies on the expression of both a small exogenous RNA and a DNA cutting enzyme. The CRISPR/Cas9 system was adapted from the prokaryotic immune system which evolved a method of identifying foreign, invading DNA from viruses and targeting it for degradation via the Cas9 enzyme3,4. This powerful genome editing technique can be used for creating targeted deletions, insertions, and mutations; and the following protocol will outline how to make deletions in a gene of interest in order to knockout its expression in vivo. The Cas9 enzyme must be expressed with a guide RNA homologous to the region of interest and contiguous with a scaffold RNA. Knockout of a gene using this technique requires targeting Cas9 to a specific region in the genome using synthetic guide RNAs (sgRNA), and inducing double stranded breaks (DSBs) at a site of interest. These DSBs are then repaired by the endogenous cell-repair machinery via non-homologous end-joining (NHEJ) which lead to indels that may produce missense or nonsense mutations and can therefore create a loss of functional protein expression5. Because this system produces genomic alterations, it only requires the transient expression of the Cas9 and sgRNA. However, it is desirable for a stable fluorescent indicator to identify cells and their progeny manipulated in this manner.
Lenti- and retroviruses have the advantage of stably integrating DNA of interest into host cells which maintain long-term expression and are passed down to daughter cells during mitosis. This protocol describes the design and production of two types of replication defective, high titer retroviruses: the human immunodeficiency virus derived lentiviral particles (lentivirus) and those based on murine Maloney Leukemia virus (retrovirus). While both of these viruses are capable of supporting stable expressing of large transgenes, the retroviral particles can only integrate into the genome during cell division with the degradation of the nuclear envelope, and therefore can be used as a tool to label and birth-date cells6. While lentiviruses have a reputation for being relatively low titer7, this methodology, including the use of caffeine8 during viral collection, routinely produces titers of 109 and 1010 particles/ml. Another advantage of lenti- and retroviruses is the tolerance for very large inserts. The following collection of protocols outlines the procedure for designing a lenti or retrovirus encoding a fluorescent reporter, sgRNAs, and Cas9 to utilize the CRISPR/Cas9 system to modify DNA as well as express a fluorescent protein.
Mouse stereotaxic neurosurgery is a valuable method for injecting viruses in vivo to study morphology, function, and connectivity of infected neurons. Viral infection in neurons can be used to manipulate expression levels over an extended period of time, such as throughout development, and expression can be precisely controlled by the use of various drug inducible systems and specific Cre driven expression. This particular protocol explains how to inject a virus expressing an sgRNA and Cas9 to knockout a gene of interest in the brain of an adult mouse. Mice recover very quickly from this procedure and expression of the viral transgene can be seen within 48 hours post injection. However, fluorophore expression appears to increase over the course of weeks resulting in near maximal levels by 3 weeks post-infection. Mice that undergo viral stereotaxic injection can be used for behavior, electrophysiology, or morphological studies. Overall, the purpose of these procedures is to demonstrate how to knockout a gene in the adult mouse brain using stereotaxic surgery and a virus expressing a specific sgRNA and Cas9.

<table><tbody><tr><td colspan="4">&lt;strong&gt;List of Cell Culture Reagents&lt;/strong&gt;</td></tr><tr><td>293FT cell line</td><td>Life Technologies</td><td>R700-07</td><td>For Lentivirus</td></tr><tr><td>293GP cell line</td><td>Clontech</td><td>631458</td><td>For Retrovirus</td></tr><tr><td>Iscove&#039;s Modification of DMEM (IMDM)</td><td>Corning&amp;nbsp;</td><td>10-016-CV</td><td>Complete IMDM with 10% FBS, 1% NEAA, 1% L-Gln, and&amp;nbsp; 1% P/S</td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>Corning</td><td>35-011-CV</td><td></td></tr><tr><td>MEM Nonessential Amino Acids (NEAA)</td><td>Corning</td><td>25-025-CI</td><td></td></tr><tr><td>L-Glutamine solution, 100x (L-Gln)</td><td>Corning</td><td>25-005-CI</td><td></td></tr><tr><td>Pennicillin/Streptomycin solution, 100x (P/S)</td><td>Corning</td><td>30-002-CI</td><td></td></tr><tr><td>Polystyrene 10&amp;nbsp;cm plate</td><td>USA Scientific</td><td>CC7682-3394</td><td></td></tr><tr><td>Trypsin EDTA 1x</td><td>Corning</td><td>25-053-CI</td><td></td></tr><tr><td colspan="4">&lt;strong&gt;List of Transfection Reagents&lt;/strong&gt;</td></tr><tr><td>5&amp;nbsp;ml polystyrene tubes</td><td>Fisher Scientific</td><td>352054</td><td></td></tr><tr><td>Calcium Chloride Dihydrate (CaCl&lt;sub&gt;2&lt;/sub&gt;)</td><td>Fisher Scientific&amp;nbsp;</td><td>C69-500</td><td>Make a 2.5 M solution in ddH&lt;sub&gt;2&lt;/sub&gt;O</td></tr><tr><td>Sodium Chloride (NaCl)</td><td>Fisher Scientific&amp;nbsp;</td><td>S271-3</td><td></td></tr><tr><td>HEPES</td><td>Fisher Scientific&amp;nbsp;</td><td>BP2939-100</td><td></td></tr><tr><td>Sodium phosphate dibasic (Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;)</td><td>Fisher Scientific&amp;nbsp;</td><td>S369-500</td><td></td></tr><tr><td>2x HEPES Buffered Saline (HBS)</td><td></td><td></td><td>500 ml: 8.2 g NaCl, 5.95&amp;nbsp;g HEPES, 0.106 g Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;, pH 7.01 (exact!)</td></tr><tr><td>Caffeine</td><td>Sigma-Aldrich</td><td>C0750-5G</td><td></td></tr><tr><td>0.22 &amp;micro;M syringe filter unit</td><td>EMD Millipore&amp;nbsp;</td><td>SLGV033RS</td><td></td></tr><tr><td>0.45 &amp;micro;M syringe filter unit</td><td>EMD Millipore&amp;nbsp;</td><td>SLHP033RS</td><td></td></tr><tr><td>60 cc L/L Syringe</td><td>Med-Vet International</td><td>MV60CCLL</td><td></td></tr><tr><td>50 ml Conical Tube</td><td>Corning</td><td>352098</td><td></td></tr><tr><td>polyethylene glycol&amp;nbsp;&amp;nbsp; 6000 (PEG 6000)</td><td>Millipore</td><td>528877</td><td></td></tr><tr><td>(10x) Phosphate Buffered Saline (PBS)</td><td>National Diagnostics</td><td>CL-253</td><td></td></tr><tr><td>0.5 ml microcentrifuge tubes</td><td>USA Scientific</td><td>1605-0000</td><td></td></tr><tr><td>Matrigel</td><td>Fisher Scientific</td><td>CB-40230A</td><td></td></tr><tr><td>6-well plate</td><td>Fisher Scientific</td><td>353046</td><td></td></tr><tr><td>Paraformaldehyde</td><td>Fisher Scientific</td><td>AC41678-5000</td><td></td></tr><tr><td>Donor Horse Serum</td><td>Cellgro</td><td>35-030-CV</td><td></td></tr><tr><td>TritonX-100</td><td>Sigma-Aldrich</td><td>X100-500ML</td><td></td></tr><tr><td>10 ml serological pipette</td><td>Fisher Scientific</td><td>357551</td><td></td></tr><tr><td>anti-GFP, rabbit, 488 conjugate</td><td>Invitrogen</td><td>A21311</td><td></td></tr><tr><td colspan="4">&lt;strong&gt;Stereotaxic Surgery Reagents&lt;/strong&gt;</td></tr><tr><td>Vet-Syringe vet use T.B. Syringe only 1 cc Luer slip T.B. 100/bx</td><td>Med-Vet International</td><td>1CCVLS</td><td></td></tr><tr><td>Isoflurane</td><td></td><td></td><td></td></tr><tr><td>Stainless Steel Scalpel Blades, #10, 100-pk</td><td>Med-Vet International</td><td>JOR580S</td><td></td></tr><tr><td>artificial tear ointment</td><td>Med-Vet International</td><td>RXPARALUBE-O</td><td></td></tr><tr><td>PVP PrepSolution&amp;nbsp;</td><td>Med-Vet International</td><td>HPIV108208H</td><td></td></tr><tr><td>normal saline&amp;nbsp;</td><td>Med-Vet International</td><td>DYND500MLSH</td><td></td></tr><tr><td>cotton tipped applicators</td><td>Med-Vet International</td><td>CTA6</td><td></td></tr><tr><td>Triple antibiotic ointment&amp;nbsp;</td><td>Med-Vet International</td><td>RXTRIP-OI15</td><td></td></tr><tr><td>MONOJECT&amp;reg; Needles Soft Pack 25 g x 5/8&quot;</td><td>Med-Vet International</td><td>25058</td><td></td></tr><tr><td>6-0 silk sutures</td><td>Med-Vet International</td><td>MV-711</td><td></td></tr></tbody></table>

designing, packaging, and delivery of high titer crispr retro and lentiviruses via stereotaxic injection

Replication defective lentiviruses or retroviruses are capable of stably integrating transgenes into the genome of an infected host cell. This technique has been widely used to encode fluorescent proteins, opto- or chemo-genetic controllers of cell activity, or heterologous expression of human genes in model organisms. These viruses have also successfully been used to deliver recombinases to relevant target sites in transgenic animals, or even deliver small hairpin or micro RNAs in order to manipulate gene expression. While these techniques have been fruitful, they rely on transgenic animals (recombinases) or frequently lack high efficacy and specificity (shRNA/miRNA). In contrast, the CRISPR/Cas system uses an exogenous Cas nuclease which targets specific sites in an organism's genome via an exogenous guide RNA in order to induce double stranded breaks in DNA. These breaks are then repaired by non-homologous end joining (NHEJ), producing insertion and deletion (indel) mutations that can result in deleterious missense or nonsense mutations. This manuscript provides detailed methods for the design, production, injection, and validation of single lenti/retro virus particles that can stably transduce neurons to express a fluorescent reporter, Cas9, and sgRNAs to knockout genes in a model organism.

Following the &#34;Protocol for Designing a Guide Strand (sgRNA) for CRISPR/Cas9 Retrovirus&#34;, oligos targeting a particular sequence are inserted into the PXL cloning vector downstream of the hU6 promoter and downstream of a guide RNA scaffold using the BbsI cloning sites (Figure 1, step 1). This sgRNA is then excised from PXL and inserted into the pRubi backbone using the BstBI and PacI sites (Figure 1, Step 2). Finally, another sgRNA cloned into PXL can be placed downstream of the first guide in pRubi-Guide1 (Figure 1, Step 3), targeting another area of the gene and increasing the chances of a knockout via NHEJ. Verification of the correct constructs should be determined by sequence analysis. Once this construct is made, it can be packaged into a virus following the &#34;Protocol for Retro/Lentiviral Production- CaPO4 Method&#34;. Successful packaging is confirmed by infection of virus into HEK293 cells in order to titer the virus. If there is no fluorophore expression then there was likely an error during packaging of the virus.
Figure 2 is a representative result of 2 retroviruses, one expressing GFP and the other expressing mCherry, co-injected into the dentate gyrus of neonatal mouse (7 days-old) and imaged 21 days post-injection. Labeling of neurons with mCherry or GFP allows morphological assessment of various genetic manipulations in the same tissue, where one virus can express a CRISPR/Cas9 mediated KO and the other a control virus, expressing solely a fluorophore. Stereotaxic injection allows precise anatomical selectivity as demonstrated by the discrete infection of the intended coordinates, the dentate gyrus. When analyzing brain sections for infection, it is important to keep surrounding tissue until it is determined that the correct anatomical region was infected. If there is no sign of infection, then it is possible that the injection occurred in a neighboring region and can be identified in surrounding sections. It can also be helpful to locate the needle track to find the exact injection region. VSVg pseudotyped viruses rarely spread out of the margins of the dentate gyrus when injected in vivo, and tend to spread along the rostral/caudal axis infecting cells along the entire dentate gyrus, as analyzed by 3D reconstruction (Figure 3).
<img alt="Figure 1" src="/files/ftp_upload/53783/53783fig1.jpg" /> 
Figure 1: Cloning strategy for the retroviral pRubi-Guide1-Guide2-CRISPR plasmid. This strategy is identical for the FU-based lentiviral plasmids. sgRNA oligos are annealed and inserted into PXL using the BsbI cloning sites. After sequencing to ensure that the sgRNA is successfully inserted into PXL, digest the plasmid with BstBI and PacI. The insert that is dropped out (black box) is then cloned into the viral back bone (black dotted lines) pRubi-Guide1 CRISPR. A second sgRNA can also be inserted into PXL and digested out using the PacI enzyme. This is then cloned into the pRubi-Guide1 CRISPR vector (red dotted lines) using the PacI site. The resultant plasmid then contains both guide strands as well as the necessary viral elements, promoters, and fluorophores. <a href="https://www.jove.com/files/ftp_upload/53783/53783fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/53783/53783fig2.jpg" /> 
Figure 2: Retroviral injection of the mouse dentate gyrus. Retroviruses expressing mCherry (red) or GFP (green) were injected into the dentate gyrus of a p7 mouse. 21 days later the mice were perfused and the brains sectioned and stained for GFP and mCherry. (A) A 5X wide-field fluorescent image shows the precision of the dentate gyrus injection and the specificity of labeling dentate gyrus granule neurons. The morphology of the hippocampus can be seen via the Dapi (blue) staining. Scale bar measures 200 &#181;m. (B) A 10X wide-field fluorescent image demonstrates that these high titer lentiviruses infect a large number of cells whose morphology can be accessed via fluorophore expression. Scale bar measures 100 &#181;m. (C) Viruses expressing GFP or mCherry were co-injected into the dentate gyrus. Using a system of retroviruses, one can use one virus to make one genetic manipulation marked by GFP and another manipulation marked by mCherry, and then assess single or additive changes due to each virus. Scale bar measures 10 &#181;m.
<img alt="Figure 3" src="/files/ftp_upload/53783/53783fig3.jpg" /> 
Figure 3: Anatomical spread of lentiviral injection. Stereotaxic coinjection of a GFP-shPten virus and an mCherry control virus into the brain of an adult Ptenloxp/+ mouse resulted in a viral spread along the entire rostral/caudal axis of the dentate gyrus of the hippocampus. This is shown in a 3D reconstruction of the extent of the injection in which closed contours of the viral spread were traced over 21 serial sections (Z = 50 &#956;m/section) using reconstruction software. The contour tracings were then aligned to generate the 3D images for volume quantification. Total viral spread is shown in green (volume = 54,730,800 &#956;m3) and dentate-localized spread is shown in purple (volume = 27,275,200 &#956;m3). The virus spreads along the needle track and the corpus callosum at the intersection of the needle track in addition to filling the rostral/caudal axis of the dentate gyrus. Scale bar measures 200 &#181;m.
<img alt="Supplemental Video 1 Frame" src="/files/ftp_upload/53783/53783video1frame.jpg" /> 
Supplemental Video 1. Design of sgRNAs to clone into retroviral and lentiviral backbone. 
In this synthetic guide RNA (sgRNA) design example, the genomic sequence of mouse CHD8 is downloaded from NCBI.&#160; The start codon and exon structure are then visualized in Vector NTI.&#160; This allows us to copy the genomic region around the first coding exon and enter this sequence into Benchling.&#160; Benchling allows us to visualize all potential sgRNAs in the region.&#160; Further, after indicating the genomic region we have input, Benchling will show us the on-target and off-target scores for each guide RNA.&#160; The user can then select the guide RNA with the highest on- and off-target scores. <a href="https://www.jove.com/files/ftp_upload/53783/Benchling_GuideRNA_Design.mp4" target="_blank">Please click here to view this video. (Right-click to download.)</a>

Watch this Scientific Journal Video about Designing, Packaging, and Delivery of High Titer CRISPR Retro and Lentiviruses via Stereotaxic Injection at JoVE.com

Designing, Packaging, and Delivery of High Titer CRISPR Retro and Lentiviruses via Stereotaxic Injection

The CRISPR/Cas9 system offers the potential to make targeted genome editing accessible and affordable to the scientific community. This protocol is intended to demonstrate how to create viruses that will knockout a gene of interest using the CRISPR/Cas9 system, and then inject them stereotaxically into the adult mouse brain.

Replication defective lentiviruses or retroviruses are capable of stably integrating transgenes into the genome of an infected host cell. This technique ...

designing-packaging-delivery-high-titer-crispr-retro-lentiviruses-via

Research

JoVE Journal

Neuroscience

17.5K Views.  Geisel School of Medicine at Dartmouth College. The CRISPR/Cas9 system offers the potential to make targeted genome editing accessible and affordable to the scientific community. This protocol is intended to demonstrate how to create viruses that will knockout a gene of interest using the CRISPR/Cas9 system, and then inject them stereotaxically into the adult mouse brain.

Video: Designing, Packaging, and Delivery of High Titer CRISPR Retro and Lentiviruses via Stereotaxic Injection

Scalable Fluidic Injector Arrays for Viral Targeting of Intact 3-D Brain Circuits

Our understanding of neural circuits--how they mediate the computations that subserve sensation, thought, emotion, and action, and how they are corrupted in neurological and psychiatric disorders--would be greatly facilitated by a technology for rapidly targeting genes to complex 3-dimensional neural circuits, enabling fast creation of "circuit-level transgenics." We have recently developed methods in which viruses encoding for light-sensitive proteins can sensitize specific cell types to millisecond-timescale activation and silencing in the intact brain. We here present the design and implementation of an injector array capable of delivering viruses (or other fluids) to dozens of defined points within the 3-dimensional structure of the brain (Figure. 1A, 1B). The injector array comprises one or more displacement pumps that each drive a set of syringes, each of which feeds into a polyimide/fused-silica capillary via a high-pressure-tolerant connector. The capillaries are sized, and then inserted into, desired locations specified by custom-milling a stereotactic positioning board, thus allowing viruses or other reagents to be delivered to the desired set of brain regions. To use the device, the surgeon first fills the fluidic subsystem entirely with oil, backfills the capillaries with the virus, inserts the device into the brain, and infuses reagents slowly (&lt;0.1 microliters/min). The parallel nature of the injector array facilitates rapid, accurate, and robust labeling of entire neural circuits with viral payloads such as optical sensitizers to enable light-activation and silencing of defined brain circuits. Along with other technologies, such as optical fiber arrays for light delivery to desired sets of brain regions, we hope to create a toolbox that enables the systematic probing of causal neural functions in the intact brain. This technology may not only open up such systematic approaches to circuit-focused neuroscience in mammals, and facilitate labeling of brain regions in large animals such as non-human primates, but may also open up a clinical translational path for cell-specific optical control prosthetics, whose precision may enable improved treatment of intractable brain disorders. Finally, such devices as described here may facilitate precisely-timed fluidic delivery of other payloads, such as stem cells and pharmacological agents, to 3-dimensional structures, in an easily user-customizable fashion.

Controlling and analyzing neural circuits in vivo would be facilitated by a technology for delivery of viruses and other reagents to desired 3-dimensional sets of brain regions. We demonstrate customized fluidic injector array fabrication, and delivery of virally-encoded optical sensitizers, enabling optical manipulation of complex brain circuits.

Our understanding of neural circuits--how they mediate the computations that subserve sensation, thought, emotion, and action, and how they are corrupted ...

Stereotactic Injection of MicroRNA-expressing Lentiviruses to the Mouse Hippocampus CA1 Region and Assessment of the Behavioral Outcome

MicroRNAs (miRNAs) are small regulatory single-stranded RNA molecules around 22 nucleotides long that may each target numerous mRNA transcripts and dim an entire gene expression pathway by inducing destruction and/or inhibiting translation of these targets. Several miRNAs play key roles in maintaining neuronal structure and function and in higher-level brain functions, and methods are sought for manipulating their levels for exploring these functions. Here, we present a direct in vivo method for examining the cognitive consequences of enforced miRNAs excess in mice by stereotactic injection of miRNA-encoding virus particles. Specifically, the current protocol involves injection into the hippocampal CA1 region, which contributes to mammalian memory consolidation, learning, and stress responses, and offers a convenient injection site. The coordinates are measured according to the mouse bregma and virus perfusion is digitally controlled and kept very slow. After injection, the surgery wound is sealed and the animals recover. Lentiviruses encoding silencers of the corresponding mRNA targets serve to implicate the specific miRNA/target interaction responsible for the observed effect, with na&iuml;ve mice, mice injected with saline and mice injected with "empty" lentivirus vectors as controls. One month post-injection, the animals are examined in the Morris Water Maze (MWM) for assessing their navigation learning and memory abilities. The MWM is a round tank filled with colored water with a small platform submerged 1 cm below the water surface. Steady visual cues around the tank allow for spatial navigation (sound and the earth's magnetic field may also assist the animals in navigating). Video camera monitoring enables measuring the route of swim and the time to find and amount the platform. The mouse is first taught that mounting the hidden platform offers an escape from the enforced swimming; it is then tested for using this escape and finally, the platform is removed and probe tests examine if the mouse remembers its previous location. Repeated tests over several consecutive days highlight improved performance of tested mice at shorter latencies to find and mount the platform, and as more direct routes to reach the platform or its location. Failure to show such improvement represents impaired learning and memory and/or anxiety, which may then be tested specifically (e.g. in the elevated plus maze). This approach enables validation of specific miRNAs and target transcripts in the studied cognitive and/or stress-related processes.

MicroRNAs have significant roles in brain structure and function. Here we describe a method to enforce hippocampal miRNA over-expression using stereotactic injection of an engineered miRNA-expressing lentivirus. This approach can serve as a relatively rapid way to assess the in vivo effects of over-expressed miRNAs in specific brain regions.

MicroRNAs (miRNAs) are small regulatory single-stranded RNA molecules around 22 nucleotides long that may each target numerous mRNA transcripts and dim an ...

Behavior

Applying Stereotactic Injection Technique to Study Genetic Effects on Animal Behaviors

Stereotactic injection is a useful technique to deliver high titer lentiviruses to targeted brain areas in mice. Lentiviruses can either overexpress or knockdown gene expression in a relatively focused region without significant damage to the brain tissue. After recovery, the injected mouse can be tested on various behavioral tasks such as the Open Field Test (OFT) and the Forced Swim Test (FST). The OFT is designed to assess locomotion and the anxious phenotype in mice by measuring the amount of time that a mouse spends in the center of a novel open field. A more anxious mouse will spend significantly less time in the center of the novel field compared to controls. The FST assesses the anti-depressive phenotype by quantifying the amount of time that mice spend immobile when placed into a bucket of water. A mouse with an anti-depressive phenotype will spend significantly less time immobile compared to control animals. The goal of this protocol is to use the stereotactic injection of a lentivirus in conjunction with behavioral tests to assess how genetic factors modulate animal behaviors.

Stereotactic injection of lentiviruses expressing cDNAs or shRNAs can modulate gene expression in specific brain areas of mice. Here, we present a protocol to combine stereotactic injections with behavioral tasks, such as the Open Field Test (OFT) and the Forced Swim Test (FST).

Stereotactic injection is a useful technique to deliver high titer lentiviruses to targeted brain areas in mice. Lentiviruses can either overexpress or ...

Packaging HIV- or FIV-based Lentivector Expression Constructs &amp; Transduction of VSV-G Pseudotyped Viral Particles

As with standard plasmid vectors, it is possible to transfect lentivectors in plasmid form into cells with low-to-medium efficiency to obtain transient expression of effectors. Packaging lentiviral expression constructs into pseudoviral particles, however, enables up to 100% transduction, even with difficult-to-transfect cells, such as primary, stem, and differentiated cells. Moreover, the lentiviral delivery does not produce the specific cellular responses typically associated with chemical transfections, such as cell death resulting from toxicity of the transfection reagent 1, 2. When transduced into target cells, the lentiviral construct integrates into genomic DNA and provides stable expression of the small hairpin RNA (shRNA), cDNA, microRNA or reporter gene 3, 4. Target cells stably expressing the effector molecule can be isolated using a selectable marker contained in the expression vector construct such as puromycin or GFP. After pseudoviral particles infect target cells, they cannot replicate within target cells because the viral structural genes are absent and the long terminal repeats (LTRs) are designed to be self-inactivating upon transduction 5, 6.
There are three main components necessary for efficient lentiviral packaging 1, 5, 6, 7. 
1. The lentiviral expression vector that contains some of the genetic elements required for packaging, stable integration of the viral expression construct into genomic DNA, and expression of the effector or reporter. 
2. The lentiviral packaging plasmids that provide the proteins essential for transcription and packaging of an RNA copy of the expression construct into recombinant pseudoviral particles. This protocol uses the pPACK plasmids (SBI) that encode for gag, pol, and rev from the HIV or FIV genome and Vesicular Stomatitis Virus g protein (VSV-G) for the viral coat protein. 
3. 293TN producer cells (derived from HEK293 cells) that express the SV40 large T antigen, which is required for high-titer lentiviral production and a neomycin resistance gene, useful for reselecting the cells for maintenance.
An overview of the viral production protocol can be seen in Figure 1. Viral production starts by co-transfecting 293TN producer cells with the lentiviral expression vector and the packaging plasmids. Viral particles are secreted into the media. After 48-72 hours the cell culture media is harvested. Cellular debris is removed from the cell culture media, and the viral particles are precipitated by centrifugation with PEG-it for concentration. Produced lentiviral particles are then titered and can be used to transduce target cells. Details of viral titering are not included in this protocol, but can be found at:
<a href="http://www.systembio.com/downloads/global_titer_kit_web_090710.pdf" target="_blank">http://www.systembio.com/downloads/global_titer_kit_web_090710.pdf</a> 8.
This protocol has been optimized using the specific products indicated. Other reagents may be substituted, but the same results cannot be guaranteed.

Packaging HIV- or FIV-based Lentivector Expression Constructs & Transduction of VSV-G Pseudotyped Viral Particles

Lentiviral expression vectors are the most effective vehicles for stably expressing different effector molecules or reporter constructs in dividing and non-dividing mammalian cells and whole organisms. Here we provide a protocol on how to package lentivector expression constructs in pseudoviral particles and to transduce target cells using the pseudoviral particles.

As with standard plasmid vectors, it is possible to transfect lentivectors in plasmid form into cells with low-to-medium efficiency to obtain transient ...

Immunology and Infection

Lentiviral Vector Platform for the Efficient Delivery of Epigenome-editing Tools into Human Induced Pluripotent Stem Cell-derived Disease Models

The use of hiPSC-derived cells represents a valuable approach to study human neurodegenerative diseases. Here, we describe an optimized protocol for the differentiation of hiPSCs derived from a patient with the triplication of the alpha-synuclein gene (SNCA) locus into Parkinson&#8217;s disease (PD)-relevant dopaminergic neuronal populations. Accumulating evidence has shown that high levels of SNCA are causative for the development of PD. Recognizing the unmet need to establish novel therapeutic approaches for PD, especially those targeting the regulation of SNCA expression, we recently developed a CRISPR/dCas9-DNA-methylation-based system to epigenetically modulate SNCA transcription by enriching methylation levels at the SNCA intron 1 regulatory region. To deliver the system, consisting of a dead (deactivated) version of Cas9 (dCas9) fused with the catalytic domain of the DNA methyltransferase enzyme 3A (DNMT3A), a lentiviral vector is used. This system is applied to cells with the triplication of the SNCA locus and reduces the SNCA-mRNA and protein levels by about 30% through the targeted DNA methylation of SNCA intron 1. The fine-tuned downregulation of the SNCA levels rescues disease-related cellular phenotypes. In the current protocol, we aim to describe a step-by-step procedure for differentiating hiPSCs into neural progenitor cells (NPCs) and the establishment and validation of pyrosequencing assays for the evaluation of the methylation profile in the SNCA intron 1. To outline in more detail the lentivirus-CRISPR/dCas9 system used in these experiments, this protocol describes how to produce, purify, and concentrate lentiviral vectors and to highlight their suitability for epigenome- and genome-editing applications using hiPSCs and NPCs. The protocol is easily adaptable and can be used to produce high titer lentiviruses for in vitro and in vivo applications.

Targeted DNA epigenome editing represents a powerful therapeutic approach. This protocol describes the production, purification, and concentration of all-in-one lentiviral vectors harboring the CRISPR-dCas9-DNMT3A transgene for epigenome-editing applications in human induced pluripotent stem cell (hiPSC)-derived neurons.

The use of hiPSC-derived cells represents a valuable approach to study human neurodegenerative diseases. Here, we describe an optimized protocol for the ...

Genetics

Selective Viral Transduction of Adult-born Olfactory Neurons for Chronic in vivo Optogenetic Stimulation

Local interneurons are continuously regenerated in the olfactory bulb of adult rodents1-3. In this process, called adult neurogenesis, neural stem cells in the walls of the lateral ventricle give rise to neuroblasts that migrate for several millimeters along the rostral migratory stream (RMS) to reach and incorporate into the olfactory bulb. To study the different steps and the impact of adult-born neuron integration into preexisting olfactory circuits, it is necessary to selectively label and manipulate the activity of this specific population of neurons. The recent development of optogenetic technologies offers the opportunity to use light to precisely activate this specific cohort of neurons without affecting surrounding neurons4,5. Here, we present a series of procedures to virally express Channelrhodopsin2(ChR2)-YFP in a temporally restricted cohort of neuroblasts in the RMS before they reach the olfactory bulb and become adult-born neurons. In addition, we show how to implant and calibrate a miniature LED for chronic in vivo stimulation of ChR2-expressing neurons.

Selective Viral Transduction of Adult-born Olfactory Neurons for Chronic in vivo Optogenetic Stimulation

Adult-born neurons of the olfactory bulb can be optogenetically controlled using Channelrhodopsin2-expressing lentiviral injection in the rostral migratory stream and chronic photostimulation with an implanted miniature LED.

Local interneurons are continuously regenerated in the olfactory bulb of adult rodents1-3. In this process, called adult neurogenesis, neural stem cells ...

Expansion of Embryonic and Adult Neural Stem Cells by In Utero Electroporation or Viral Stereotaxic Injection

Somatic stem cells can divide to generate additional stem cells (expansion) or more differentiated cell types (differentiation), which is fundamental for tissue formation during embryonic development and tissue homeostasis during adulthood 1. Currently, great efforts are invested towards controlling the switch of somatic stem cells from expansion to differentiation because this is thought to be fundamental for developing novel strategies for regenerative medicine 1,2. However, a major challenge in the study and use of somatic stem cell is that their expansion has been proven very difficult to control.
Here we describe a system that allows the control of neural stem/progenitor cell (altogether referred to as NSC) expansion in the mouse embryonic cortex or the adult hippocampus by manipulating the expression of the cdk4/cyclinD1 complex, a major regulator of the G1 phase of the cell cycle and somatic stem cell differentiation 3,4. Specifically, two different approaches are described by which the cdk4/cyclinD1 complex is overexpressed in NSC in vivo. By the first approach, overexpression of the cell cycle regulators is obtained by injecting plasmids encoding for cdk4/cyclinD1 in the lumen of the mouse telencephalon followed by in utero electroporation to deliver them to NSC of the lateral cortex, thus, triggering episomal expression of the transgenes 5-8. By the second approach, highly concentrated HIV-derived viruses are stereotaxically injected in the dentate gyrus of the adult mouse hippocampus, thus, triggering constitutive expression of the cell cycle regulators after integration of the viral construct in the genome of infected cells 9. Both approaches, whose basic principles were already described by other video protocols 10-14, were here optimized to i) reduce tissue damage, ii) target wide portions of very specific brain regions, iii) obtain high numbers of manipulated cells within each region, and iv) trigger high expression levels of the transgenes within each cell. Transient overexpression of the transgenes using the two approaches is obtained by different means i.e. by natural dilution of the electroporated plasmids due to cell division or tamoxifen administration in Cre-expressing NSC infected with viruses carrying cdk4/cyclinD1 flanked by loxP sites, respectively 9,15.
These methods provide a very powerful platform to acutely and tissue-specifically manipulate the expression of any gene in the mouse brain. In particular, by manipulating the expression of the cdk4/cyclinD1 complex, our system allows the temporal control of NSC expansion and their switch to differentiation, thus, ultimately increasing the number of neurons generated in the mammalian brain. Our approach may be critically important for basic research and using somatic stem cells for therapy of the mammalian central nervous system while providing a better understanding of i) stem cell contribution to tissue formation during development, ii) tissue homeostasis during adulthood, iii) the role of adult neurogenesis in cognitive functions, and perhaps, iv) better using somatic stem cells in models of neurodegenerative diseases.

Expansion of Embryonic and Adult Neural Stem Cells by In Utero Electroporation or Viral Stereotaxic Injection

Controlling the expansion of somatic stem cells is a major factor hampering their study and use in therapy. Here we describe a system to temporally control neural stem cells expansion during development and adulthood, which can be used to increase the number of neurons generated in the mouse brain.

Somatic stem cells can divide to generate additional stem cells (expansion) or more differentiated cell types (differentiation), which is fundamental for ...

A Method for High Fidelity Optogenetic Control of Individual Pyramidal Neurons In vivo

Optogenetic methods have emerged as a powerful tool for elucidating neural circuit activity underlying a diverse set of behaviors across a broad range of species. Optogenetic tools of microbial origin consist of light-sensitive membrane proteins that are able to activate (e.g., channelrhodopsin-2, ChR2) or silence (e.g., halorhodopsin, NpHR) neural activity ingenetically-defined cell types over behaviorally-relevant timescales. We first demonstrate a simple approach for adeno-associated virus-mediated delivery of ChR2 and NpHR transgenes to the dorsal subiculum and prelimbic region of the prefrontal cortex in rat. Because ChR2 and NpHR are genetically targetable, we describe the use of this technology to control the electrical activity of specific populations of neurons (i.e., pyramidal neurons) embedded in heterogeneous tissue with high temporal precision. We describe herein the hardware, custom software user interface, and procedures that allow for simultaneous light delivery and electrical recording from transduced pyramidal neurons in an anesthetized in vivo preparation. These light-responsive tools provide the opportunity for identifying the causal contributions of different cell types to information processing and behavior.

A Method for High Fidelity Optogenetic Control of Individual Pyramidal Neurons In vivo

The recent development of neuroscience tools that combine genetics and optics, termed "optogenetics", enables control over neural circuit activity with an unprecedented level of spatial and temporal resolution. Here we provide a protocol for integrating in vivo recording with optogenetic manipulation of genetically-defined subsets of prefrontal cortical and subicular pyramidal neurons.

Optogenetic methods  have emerged as a powerful tool for elucidating neural circuit activity  underlying a diverse set of behaviors across a broad range ...

Effective Systemic Delivery in Mice Using Retrobulbar Injection Technique

Systemic Treatment for Postnatal, Juvenile, and Runted Adult Mice by Retrobulbar Sinus Injection

While tail vein injections are frequently used as a systemic route of delivery in adult mice, retrobulbar injections are an alternative method for systemic delivery with fewer limitations. First, tail vein injections (TVIs) are limited to adult mice where the size of the tail vein is suitable for access. Being restricted to treating adult mice can be problematic when dealing with mouse models that do not survive to adulthood. Second, TVIs are not feasible for mouse models with growth retardation phenotypes where the mice never achieve the size of adult wildtype mice. Therefore, retrobulbar injections can be successfully used to treat both young and small adult mice. Lastly, retrobulbar injections are performed under anesthesia, which is less stressful on the mice than TVIs that are commonly performed without anesthesia. This article presents a protocol and detailed instructions for retrobulbar injections that can be used for systemic delivery to small and young mice.

This article provides a protocol and an accompanying video for the retrobulbar sinus injection of up to a total volume of 150 &#181;L for postnatal, juvenile, and runted adult mice. This procedure is particularly well suited for the injection of small mice (15 g) when tail vein injection is not feasible.

While tail vein injections are frequently used as a systemic route of delivery in adult mice, retrobulbar injections are an alternative method for ...

Direct Injection of a Lentiviral Vector Highlights Multiple Motor Pathways in the Rat Spinal Cord

Introducing proteins of interest into cells in the nervous system is challenging due to innate biological barriers that limit access to most molecules. Injection directly into spinal cord tissue bypasses these barriers, providing access to cell bodies or synapses where molecules can be incorporated. Combining viral vector technology with this method allows for introduction of target genes into nervous tissue for the purpose of gene therapy or tract tracing. Here a virus engineered for highly efficient retrograde transport (HiRet) is introduced at the synapses of propriospinal interneurons (PNs) to encourage specific transport to neurons in the spinal cord and brainstem nuclei. Targeting PNs takes advantage of the numerous connections they receive from motor pathways such as the rubrospinal and reticulospinal tracts, as well as their interconnection with each other throughout spinal cord segments. Representative tracing using the HiRet vector with constitutively active green fluorescent protein (GFP) shows high fidelity details of cell bodies, axons and dendritic arbors in thoracic PNs and in reticulospinal neurons in the pontine reticular formation. HiRet incorporates well into brainstem pathways and PNs but shows age dependent integration into corticospinal tract neurons. In summary, spinal cord injection using viral vectors is a suitable method for introduction of proteins of interest into neurons of targeted tracts.

This protocol demonstrates injection of a retrogradely transportable viral vector into rat spinal cord tissue. The vector is taken up at the synapse and transported to the cell body of target neurons. This model is suitable for retrograde tracing of important spinal pathways or targeting cells for gene therapy applications.

Introducing proteins of interest into cells in the nervous system is challenging due to innate biological barriers that limit access to most molecules. ...

Designing, Packaging, and Delivery of High Titer CRISPR Retro and Lentiviruses via Stereotaxic Injection

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Selective Viral Transduction of Adult-born Olfactory Neurons for Chronic in vivo Optogenetic Stimulation

Expansion of Embryonic and Adult Neural Stem Cells by In Utero Electroporation or Viral Stereotaxic Injection

A Method for High Fidelity Optogenetic Control of Individual Pyramidal Neurons In vivo

Systemic Treatment for Postnatal, Juvenile, and Runted Adult Mice by Retrobulbar Sinus Injection

Direct Injection of a Lentiviral Vector Highlights Multiple Motor Pathways in the Rat Spinal Cord