Protocol for lentivirus-mediated delivery of genes to study neurogenesis and cognitive function in adult rodents

Summary Adult neurogenesis leads to the generation of functional neurons from neural stem cells, whereas impairment of adult hippocampal neurogenesis contributes to the pathophysiology of cognitive symptoms in neurodegenerative and neuropsychiatric diseases. Here, we present a protocol for a direct hippocampal injection of lentivirus-delivered gene in adult rodents to study the specific molecular mechanism underlying adult neurogenesis, including lentivirus packaging and stereotaxic injection, EdU and BrdU injections, tissue immunostaining and imaging analysis, and cognitive testing. For complete details on the use and execution of this protocol, please refer to Li et al. (2023).1


Highlights
Step-by-step guide for lentivirus production Protocol for stereotactic lentiviral injection into the dentate gyrus of rodent hippocampus

Protocol
Protocol for lentivirus-mediated delivery of genes to study neurogenesis and cognitive function in adult rodents
Note: You need to have 95%-100% confluent HEK293T in one 150 mm dish.Split 1 dish of 150 mm into 5 3 150 mm dishes (wash with DPBS and strong trypsin for 5 min).Each plate HEK293T cells should have 80% confluent at the moment of transfection.

Preparation of lentivirus stereotactic injection
Timing: $2 days 4. Make sure to sterilize all surgical instruments (a pair of scissors, blunt-end forceps, a needle holder, scalpel, cotton swabs, absorbable sutures, and an electric razor) and experimental site (a stereotactic apparatus with digital display of the stereotaxic frame, a dental drill, a 5 mL Hamilton syringe fitted with a 33-gauge needle, a syringe pump controller and the rodent anesthesia machine).
Note: Sterilize surgical instruments by autoclaving and sterilize the surgical field by UV irradiation.
5. Preparation of reagents (75% ethyl alcohol, isoflurane, analgesic (ketoprofen), erythromycin eye ointment and LVs).6. Preparation of 12-month-old C57BL/6 male or female wild-type mice (ensuring that mice are of sufficient number, of the correct sex, and of suitable physical condition for the operation).
Note: Adult C57BL/6 mice (12-month-old) are housed under standard conditions (feeding on a 12-h light/dark cycle with lights and given free access to food and water).

Preparation for EdU or BrdU solution
Timing: 1 h 7. Prepare an EdU or BrdU solution at 10 mg/mL concentration.a.The solvent is DPBS.b.Dissolve by vortexing and heat in a 42 C thermometer if necessary.c. 10 mg EdU or BrdU dissolved in 1 mL DPBS will result in the applied concentration (10 mg/mL).
Note: EdU and BrdU are recommended to be stored in powder form and protected from light at À20 C. Usually, fresh solution is required for application.

Preparation of tissue and immunostaining solutions
Timing: $2 days 8. Preparation of 0.2 M phosphate buffer (PB, see materials and equipment section, stored at 25 C for up to 1 month), 4% paraformaldehyde (PFA, see materials and equipment section, stored at 4 C for up to 2 weeks protected from light), cryoprotectant solution (see materials and equipment, stored at 4 C for up to 1 month), Click Additive Solution (according to the manufacturer's instructions (https://www.beyotime.com/product/ST067-50mg.htm) and store it at À20 C refrigerator after dispensing) and Polyvinyl alcohol (PVA)-DABCO solution(see materials and equipment, stored at À20 C for up to 6 months and 4 C for up to 1 week).

Note:
The components and storage methods are described in the Materials and Equipment section.

STEP-BY-STEP METHOD DETAILS Production of lentivirus
Timing: $1 week 1.Check for the confluence of HEK293T cells (recommend is 80%) through the microscope and replace the culture medium with 10 mL fresh culture medium 1 h before transfection.
CRITICAL: If HEK293T cells have over-grown, showing lots of cluster and floaters, discard them and start new cells.
2. Prepare the mixture of helper plasmids and target plasmid with PEI (Figure 1).
Note: Dilute total DNA (target plasmid and helper plasmid) and PEI (1 mg/mL) in 1:2.5 ratio (1 mg of DNA: 2.5 mL of PEI) a.Take one sterile 50 mL Eppendorf (EP) tube, add 5 mL of Opti-MEM I Reduced Serum Media, and add the plasmids as follows: Mix this mixture by pipette (approximately 10 times) using a 5 mL disposable pipette.Label the tube ''DNA'' and incubate for 5 min at 25 C. Note: PEI, a non-viral cationic polymer, can be used to transfect plasmid DNA or siRNA into suspension or adherent cells due to its low toxicity, low cost, and low immunogenicity. 4ternatives: You can use Lipofectamine 3000 transfection reagent to replace PEI. 5 c.Add PEI diluent to plasmid diluent 50 mL EP tube (DNA tube), gently mix by pipette (approximately 10 times) using a 10 mL disposable pipette.Incubate for 20 min at 25 C. d.Take out 5 dishes of HEK293T cell from incubator, add 2 mL the mixture of DNA-PEI drop by drop to each dish, gently mix, mark dishes, and put them back to incubator.3. Remove media by aspiration after 4-6 h transfection, add 13 mL fresh culture media (see materials and equipment) to each dish.
CRITICAL: Transfection time cannot over 6 h, if the transfection time is too long, it will affect the cell heath and survival.5. Filter virus through a 0.22 mm vacuum bottle filter and centrifuge at 49000 g, 4 C for 2 h.Discard the supernatant by aspiration (Figure 2).
Alternatives: If you do not have ultracentrifuge, you can use polyethylene glycol 6000 to concentration and purification of the virus. 6Wash virus once with cold DPBS and then perform the final centrifugation at 49000 g, 4 C for 2 h. 7. Leave approximately 100 mL of cold DPBS in the tube to resuspend the viruses' particles, and then stored at 4 C for 24 h.8. Aliquot the viruses at 10 mL per 1.5 mL tube, store at À80 C.
Note: All instruments, container, tips, tubes used for packaging LVs should be decontaminated with freshly made 10% bleach after use.Note: A minimum of $1310 9 infectious particles per ml is recommended.
For more detailed protocol of lentivirus production see Fricano-Kugler et al. and Guo et al. 6,7 Preparation for surgery of animals Timing: 1 h 10.Assemble the stereotaxic instrument, the dental drill and the rodent anesthesia machine (Figure 3A).11.Anesthetize the mouse with isoflurane by a mask.
Note: Induction with 5% gas in air mixture before transferring to the stereotactic frame, then maintenance at 2%.
12. Wait until the mouse is fully anesthetized, then inject Ketoprofen (3-5 mg/kg) via i.p. route to manage pain.
13. Place the mouse on a heating pad to keep a steady body temperature.
Note: Mice under anesthesia may have difficulties regulating body temperature, a heat pad will prevent hypothermia and unexpected animal deaths during surgical procedures.
14. Moisten the mouse eyes with ointment to prevent drying and infection.15.Use an electric razor to remove hair from the top of the head.16.Sanitize the skin with 75% ethanol.
Note: If possible, please perform a complete surgical scrub consisting on 3 independent cleaning procedures alternating Alcohol and iodine.

Exposure of the skull and drilling
Timing: 10 min 17.Latch the mouse front teeth onto the anterior clamp of the adapter on the stereotactic instrument, and adjust the rods into the crevices just anterior to the animal's ears to fix the head.Note: Not all stereotactic frames are built in digital measure tools.It requires operators have a knowledge in the use of Vernier scale in non-digital equipment.
22. Find the coordinates of the DG based on the atlas of the mouse brain 8 : À2.0 mm posterior to the bregma, G1.7 mm lateral to the midline, -1.9 mm ventral to the surface of the skull.23.Move the needle of the syringe according above coordinates, label the two positions with a marker.24.Raise the syringe in the vertical axis and move the tip to a safe position.25.Using a fine dental drill (0.6 mm drill bit) carefully grind the skull at the target site to two shallow holes (Figure 3C).
Note: It is recommended to use angled Drill position instead of vertical to avoid falling deep in to the brain tissue.If bleeding occurs during this procedure, use a small medical cotton ball to soak up the blood.
CRITICAL: Drilling holes must be properly controlled, otherwise brain tissue damage can easily be caused accidentally after drilling through the skull, which adversely affects the process and results of the experiments.

Lentivirus injection
Timing: $20 min 26.Take out the moderate consumption of LVs from -80 C refrigerator, and put it on the ice right before viral loading.
Note: Lentivirus expressing PTN are named as lenti-PTN and lentivirus without the gene of interest (e.g.: PTN) are named as lenti-NC to be prepared and used as control.
CRITICAL: Virus is stored in the freezer at À80 C. Use it immediately after removal, and avoid multiple freeze-thaw processes.
27. Clean the microinjection syringe with sterile PBS and withdraw 1 mL of the concentrated LVs solution.
Note: When connecting the needle to the micro syringe pump, ensure that the tip of the needle is vertical and that there are no air bubbles inside the needle.A good practice is to collect and withdraw volumes to remove the air.Specifically, a first collection with a Hamilton will take significant air that can be seen in the plunger end (more than 5mL).
28. Place the syringe over the hole and slowly lower it vertically until it reaches the surface of the brain.Set the dorsal/ventral (D/V) coordinate (depth) to 0. 29.Lower the syringe to reach the DG, set D/V to À1.9 mm (Figure 3D).30.Set the digital pump to 0.2 mL/min (1 mL would be injected in 5 min) and start infusion.
Note: Infusion at a slow pace allows the virus to spread efficiently into the tissues and prevents reflux.
31.After finishing the infusion, wait an additional 5 min to complete diffusion of LVs into the brain rather than backflow of viral solution up the needle track.32.Slowly remove the syringe out of the brain and watch for fluid backflow.
Note: If reflux is clearly observed, it means partial loss of LVs injection.Protocol 33.Rinse the microinjection syringe with sterile PBS again to remove traces of blood on the tip of the needle and keep the syringe is unobstructed.34.Repeat step 27-32 for another hemisphere.
For more detailed protocol of LVs injection, see Li et al. and Tang et al. 1,3 Wound sealing and post-operative care Timing: 30 min to 1 h 35.Suture the wound with absorbable thread.36.Gently remove the ear bars from the stereotaxic frame and take the mouse from the operating table.37. Place the mouse in a new clean cage and keep it on a heating pad (Figure 3E).38.Closely monitor the mouse until it wakes up and moves on its own.39.Return the mouse to the standard housing condition.
Note: Mice are required in recovery for 72 h before undergoing the next experimental step.

BrdU injection
Timing: 4 days 3 days after the operation, mice receive BrdU injection to assess NSCs differentiation.40.Treat mice with BrdU at 50 mg/kg i.p. for 4 days and sacrificed at 2 or 4 weeks after injection to assess NSCs differentiation in vivo (Figure 4C).
CRITICAL: Make fresh solution for BrdU injection each day.
Note: Take the injection at the same time each day.

EdU injection
One week after the operation, mice receive EdU injection to assess NSCs proliferation.
Note: In this study, PTN overexpressing lentivirus showed higher infection efficiency in mouse hippocampus one week after injection, so we performed proliferation assay one week later.However, the timing can be adjusted depending on the purpose of the experiment; for example, some studies have assayed NSCs proliferation 2 or 3 weeks after viral injection. 7,9.Treat mice with EdU at 100 mg/kg i.p. and then sacrificed 2 h after injection to assess NSCs proliferation in vivo (Figure 4A).
CRITICAL: Make fresh solution for injection.

Timing: $2 weeks
The behavioral assessment in the Morris Water Maze (MWM) and the Reverse Morris Water Maze (RMWM) are performed after 4 weeks, at which LVs usually infect most cells within the injection sphere.These behavior tests reflect spatial learning and memory ability of mice.The detailed protocol of MWM and RMWM see Vorhees and Williams and Chen et al. 12,13 Note: The RMWM test was conducted 48 h after the probe test of the MWM test.

EXPECTED OUTCOMES
To investigate whether pleiotrophin (PTN) affected proliferation and differentiation of NSCs, we stereotaxically injected LVs co-expressing green fluorescent protein (GFP) with PTN into the DG of hippocampus to induce PTN overexpression of the old mouse (12-month-old wild-type mouse) (Figure 4A).After one week LVs injection, we treated 5 mice of each group (PTN group and the control group) with EdU by i.p. injection 2 h before sacrificing to label proliferating cells (Figure 4A).As expected, PTN overexpression in the DG of the old mouse resulted in a significant increase in the number of GFP + EdU + GFAP + NSCs (with a typical NSC morphology) (Figure 4B), which suggests that PTN promotes the proliferation of NSCs.And we treated another cohort mice of the two groups with BrdU through i.p. injection for 4 days and sacrificed at 2 or 4 weeks after injection respectively to label immature neurons and mature neurons in vivo (Figure 4C).Similarly, the numbers of both GFP + BrdU + DCX + immature neurons (Figure 4D) and GFP + BrdU + NeuN + mature differentiated neurons (Figure 4E) were remarkably increased, suggesting that PTN induces more neuronal differentiation.Moreover, we subjected new cohort mice of the two groups to perform MWM and RMWM tests 4 weeks after injection lentiviruses to assess their cognitive function (Figure 5A).We found that either in MWM or RMWM tests, PTN group mice crossed the target quadrant more times and spent less time in the latency than the control group mice (Figures 5B-5G), indicating better spatial memory.Taken together, these results demonstrated that PTN ameliorates both age-induced hippocampal neurogenesis defects and cognitive dysfunction.

LIMITATIONS
We have evaluated transfection efficiency by detecting native fluorescence protein of infected cells.Although it is a simple and straightforward method, one possible technical limitation is transfected cells might exhibit lower sensitivity of native fluorescence detection due to cell types and transfection time.Thus, immunohistochemistry and subsequent indirect fluorescence detection is more credible.
The viral vector in our study is popular and has been used in our previous studies. 3cause each viral vector varies in dissemination capacity, packaging capacity, tropism, transgene expression time, and expression duration.Moreover, it is challenging, particularly in brain in vivo, since the quantity of infected cells in the specific tissue can be limiting.If possible, pilot studies should be performed before the formal experiment to determine the dosage of virus and validate viral function.

TROUBLESHOOTING Problem 1
Transfection efficiency is < 80% or the transfected cells are not bright fluorescent (step 1-4).

Note:
When you first use those plasmids, check your plasmid with restriction enzymes or sequence to make sure you have right plasmid (e.g.: cutting the VSV-G plasmid with restriction endonucleases HindIII and EcoRI produces three fragments of 5.4 kb, 1.6 kb and 0.58 kb in length; cutting the pMDLg plasmid with restriction endonuclease EcoRI produces three fragments of 4.3 kb, 4.1 kb and 0.4 kb in length; and cutting the pRSV-Rev plasmid with restriction endonuclease EcoRI produces two fragments of 4.1 kb and 0.3 kb in length).The concentration of plasmid also needs to be confirmed to ensure the amount added (e.g.: the original concentration of VSV-G is 800 ng/mL (measured by NanoDrop instrument), therefore the volume of VSV-G added is 50 mL (40*1000/800 = 50 mL)).The media should be stored at 25 C.The medium should be stored at À20 C for up to 6 months and 4 C for up to 1 week.another sterile 50 mL EP tube, add 5 mL of Opti-MEM I Reduced Serum Media, add 862.5 mL PEI (1 mg/mL), mix by pipette (approximately 10 times) using a 5 mL disposable pipette.Label the tube ''PEI'' and incubate for 5 min at 25 C.

4 .
Harvest released viral particles by collecting viral supernatant at 40, 64, and 88 h in a T75 flask, stored at 4 C. Note: It is important to change the media with extreme gentleness to avoid the detach from the plate.CRITICAL: Check the transfection efficiency approximately 40 h after transfection through detecting whether the transfected cells have bright florescent.If efficiency is <80%, it means that you should have not made virus out of it, stop your experiment.

Figure 1 .
Figure 1.Schematic of production of lentivirus (LVs) from transfected HEK293T cells LVs that up-regulation of PTN gene were constructed by the third-generation lentiviral vectors.Virus particles are harvested immediately after consecutive 3-day viral supernatant collection.

9 .
Measure virus titer by infecting HEK293T cells with serially diluted virus preparations.a. Plate HEK293T cells in 6-well plate (5 3 10^4 cells/per well) a day before viral titer infection.b.Prepare viruses of different titers by serial dilution (1:1, 1:10, 1:100.1:10^6).c. 2 mL different titers of virus are added separately to each well with 72-120 h incubation at 37 C. d.Wash with PBS three times.e. Add 200 mL trypsin (stored at 4 C for several months) to each well for 5 min, then collect the cells solution into a fluorescence-activated cell sorting (FACS) tube and add 600 mL 4% PFA.Note: Samples can keep stable for one week when stored at 4 C in the dark.f.Calculate viral titer from FACS result.

Figure 2 .
Figure 2. Preparation for lentivirus (LVs) harvest by ultracentrifugation (A) Main components before LVs harvest by ultracentrifugation. (B) The ultracentrifuge is used for LVs harvest.(C) The proper SW32Ti swinging-bucket rotor and the matched six tubes were well installed.(D) Choose the right rotor and laboratory consumables.(E) Set up the correct parameters in order: 20,000 rpm, 2 h, and 4 C.

TABLE REAGENT
The media should be stored at 4 C for up to 3 weeks.Fresh medium should be prepared before each differentiation experiment or kept sterile and stored at 4 C for up to 1 month.Add 0.2 M Tris-HCl, adjust the solution to pH 8-8.5.Aliquot (1 mL is recommended) and store at À20 C. Do not refreeze.
Note: Dissolve first with 800 mL ddH 2 O and then add ddH 2 O until volume reaches 1 L. Adjust pH to 7.4.Pure the solution by filtering with a 0.22 mm filter.Note: Dissolve first with 800 mL 0.2 M PB and then add 0.2 M PB until volume reaches 1 L. Aliquot (50 mL is recommended), and store at À20 C. The media should be stored at 25 C. Fresh medium should be prepared or stored at 25 C for up to 1 month.PFA should be stored at 4 C and the other media should be stored at 25 C.The medium should be stored at 4 C for up to 2 weeks protected from light.(Continued on next page) Protocol Note: