Direct Adeno-associated Viruses Injection of Murine Adipose Tissue

Materials and Reagents

1. Animals: 8–12-week-old C57BL/6JNarl mice (National Laboratory Animal Center)

2. Rodent MD’s^TM Rimadyl (Carprofen, 2 mg/tablet) (Bio-Serv, catalog number: SMD150-2)

3. AAV8 vectors carrying enhanced green fluorescent protein (GFP) as reporter gene driven by CMV promoter (obtained from National RNAi Core Facility at Academia Sinica, Taiwan)

4. PBS (Gibco, catalog number: 10010023)

5. 75% ethanol

6. FORANE^® isoflurane (Abbott, catalog number: B506)

7. Bacitracin-neomycin ointment (Shiteh, catalog number: 022990)

Equipment

1. Matrx^® anesthesia machine (Midmark, catalog number: VIP3000) (Figure 1, ①)

2. Anesthetic gas recovery machine (Step, catalog number: R-600) (Figure 1, ②)

3. Anesthetized mouse facemask (RWD, catalog number: 68635) (Figure 1, ③)

4. Surgery tweezers (Shineteh, catalog number: ST-TW011) (Figure 1, ④)

5. Surgery scissor (Shineteh, catalog number: ST-S011PK) (Figure 1, ⑤)

6. 50 µL syringe (Hamilton, catalog number: 80901); needles sold separately (model: 1705 LT) (Figure 1, ⑥)

7. Needle (30 G × ½) (BD Precision Glide^TM, catalog number: 305106) (Figure 1, ⑦)

8. Needle holder (Shineteh, catalog number: ST-H212) (Figure 1, ⑧)

9. Sterilized Suture, 4-0, 12 mm 3/8 circle (UNIK, catalog number: SC124) (Figure 1, ⑨)

10. Biosafety Cabinet (ClassII)

11. Hair clipper (Orbaner, catalog number: MB-022)

    
    

    
    Figure 1. Setup of experiment. ① Anesthesia machine, ② Anesthetic gas recovery machine, ③ Anesthetized mouse facemask, ④ Surgery tweezers, ⑤ Surgery scissor, ⑥ Syringe, ⑦ 30 G needle, ⑧ Needle holder, ⑨ Sterilized suture.

Procedure

1. Replace the diet with rodent MD’s^TM Rimadyl 16 h before surgery to prevent postoperative pain.

2. Anesthetize mice in an anesthesia chamber filled with isoflurane at a flow rate of 5% in 100% oxygen.

3. Assess the level of anesthesia by pinching the mice’s hind toes or tail end (no withdrawal should be observed).

4. Transfer mice to a biosafety cabinet and put on an anesthetized mouse facemask. Adjust the rate of isoflurane to 1.5%–2.5% in 100% oxygen.

5. Shave a small area in the flanks and proximal hip joints with a hair clipper (Figure 2A).

   Video 1 shows the procedures in steps 5–10.

   
   
   
   Video 1. Procedures of direct adeno-associated virus (AAV) injection of murine adipose tissue. The video was produced at National Yang Ming Chiao Tung University (NYCU), where all procedures followed guidelines from the NYCU and were approved by the Institutional Animal Care and Use Committee (IACUC) of NYCU (IACUC approved number: 1090706, 1090706r, 1100332).

   
   

6. Clean the shaved region with 75% ethanol.

7. Prepare an AAV8 solution in PBS. AAV8 will be administered at a total of 1 × 10¹¹–2 × 10¹¹ viral genomes (VG) AAV8 in a 30 μL volume for one fat pad per mouse. Fit a 30G needle securely with Hamilton syringe. Preload syringe with the AAV8 solution. Avoid air in the syringe.

8. Tent the skin with the tweezers and make a 0.5–1 cm incision in the skin using surgical scissors.

9. Expose the fat pad and carefully insert a 30G needle until the needle bevel enters the tissue. Inject the AAV8 slowly into 4–5 distinct spots (6–8 μL per spot) of the fat pad. Hold the needle in place for a little while after each injection to prevent backflow into the syringe (Figure 2B).

10. Hold the needle of the 4-0 braided silk with a needle holder and suture the incision (Figure 2C). (Alternative: close the incision with Michel's suture clips.)

11. Apply ointment to the incision region until it heals to prevent post-operative infection.

12. Transfer the mice to a clean cage on a heating pad. Monitor the mice closely until they regain consciousness.

13. Each experimental mouse must be single housed in a cage after surgery until the incision heals. Provide mice with Rimadyl within 48 h after the surgery to alleviate pain.

14. After one week from injection, the GFP signal is detectable.

    
    

    
    Figure 2. Direct adipose adeno-associated virus (AAV) injection procedure. A. Shave and clean the surgery area at the flank and proximal hip joints. The surgical incision region is shown as a double-headed arrow. B. Make a 0.5 cm incision and carefully inject the AAV into multiple spots in the fat pad. C. Suture the incision using 4-0 braided silk.

Notes

1. All materials used in this experiment must be sterilized or autoclaved to prevent contamination.

2. Observe the mice daily to ensure there are no surgical complications such as infection, bleeding, or poor wound healing. Apply the ointment to the incision region or prolong the Rimadyl feeding if particular surgical complications arise.

Acknowledgments

We thank the National RNAi Core Facility at Academia Sinica in Taiwan for providing AAV8 reagents and related services. This work was financially supported by the “Cancer Progression Research Center, National Yang Ming Chiao Tung University” from The Featured Areas Research Center Program within the framework of the Higher Education Sprout Project by the Ministry of Education (MOE) in Taiwan. This work was supported by the MOST 111-2740-B-A49-001 and NSTC 111-2634-F-A49-014. We have successfully applied this protocol to our current publication (Wu et al., 2022).

Competing interests

The authors declare that no competing financial interests exist.

Ethics

All the animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of National Yang Ming Chiao Tung University. (IACUC #1090706, 1090706r, 1100332) and housed on a 12:12 h light/dark cycle at 22 °C.

References

1. Atchison, R. W., Casto, B. C. and Hammon, W. M. (1965). Adenovirus-Associated Defective Virus Particles. Science 149(3685): 754-756.
2. Bates, R., Huang, W. and Cao, L. (2020). Adipose Tissue: An Emerging Target for Adeno-associated Viral Vectors. Mol Ther Methods Clin Dev 19: 236-249.
3. Cao, H., Molday, R. S. and Hu, J. (2011). Gene therapy: light is finally in the tunnel. Protein Cell 2(12): 973-989.
4. Flotte, T. R., Afione, S. A. and Zeitlin, P. L. (1994). Adeno-associated virus vector gene expression occurs in nondividing cells in the absence of vector DNA integration. Am J Respir Cell Mol Biol 11(5): 517-521.
5. Haczeyni, F., Bell-Anderson, K. S. and Farrell, G. C. (2018). Causes and mechanisms of adipocyte enlargement and adipose expansion. Obes Rev 19(3): 406-420.
6. Haslam, D. W. and James, W. P. (2005). Obesity. Lancet 366(9492): 1197-1209.
7. Jimenez, V., Munoz, S., Casana, E., Mallol, C., Elias, I., Jambrina, C., Ribera, A., Ferre, T., Franckhauser, S. and Bosch, F. (2013). In vivo adeno-associated viral vector-mediated genetic engineering of white and brown adipose tissue in adult mice. Diabetes 62(12): 4012-4022.
8. Jo, J., Gavrilova, O., Pack, S., Jou, W., Mullen, S., Sumner, A. E., Cushman, S. W. and Periwal, V. (2009). Hypertrophy and/or Hyperplasia: Dynamics of Adipose Tissue Growth. PLoS Comput Biol 5(3): e1000324.
9. O'Neill, S. M., Hinkle, C., Chen, S. J., Sandhu, A., Hovhannisyan, R., Stephan, S., Lagor, W. R., Ahima, R. S., Johnston, J. C. and Reilly, M. P. (2014). Targeting adipose tissue via systemic gene therapy. Gene Ther 21(7): 653-661.
10. Pipe, S., Leebeek, F. W. G., Ferreira, V., Sawyer, E. K. and Pasi, J. (2019). Clinical Considerations for Capsid Choice in the Development of Liver-Targeted AAV-Based Gene Transfer. Mol Ther Methods Clin Dev 15: 170-178.
11. Salans, L. B., Cushman, S. W. and Weismann, R. E. (1973). Studies of human adipose tissue. Adipose cell size and number in nonobese and obese patients. J Clin Invest 52(4): 929-941.
12. Spalding, K. L., Arner, E., Westermark, P. O., Bernard, S., Buchholz, B. A., Bergmann, O., Blomqvist, L., Hoffstedt, J., Naslund, E., Britton, T., et al. (2008). Dynamics of fat cell turnover in humans. Nature 453(7196): 783-787.
13. Swinburn, B. A., Sacks, G., Hall, K. D., McPherson, K., Finegood, D. T., Moodie, M. L. and Gortmaker, S. L. (2011). The global obesity pandemic: shaped by global drivers and local environments. Lancet 378(9793): 804-814.
14. Uhrig-Schmidt, S., Geiger, M., Luippold, G., Birk, G., Mennerich, D., Neubauer, H., Grimm, D., Wolfrum, C. and Kreuz, S. (2014). Gene delivery to adipose tissue using transcriptionally targeted rAAV8 vectors. PLoS One 9(12): e116288.
15. Vishvanath, L. and Gupta, R. K. (2019). Contribution of adipogenesis to healthy adipose tissue expansion in obesity. J Clin Invest 129(10): 4022-4031.
16. Wabitsch, M., Funcke, J. B., von Schnurbein, J., Denzer, F., Lahr, G., Mazen, I., El-Gammal, M., Denzer, C., Moss, A., Debatin, K. M., et al. (2015). Severe Early-Onset Obesity Due to Bioinactive Leptin Caused by a p.N103K Mutation in the Leptin Gene. J Clin Endocrinol Metab 100(9): 3227-3230.
17. Wang, D., Tai, P. W. L. and Gao, G. (2019). Adeno-associated virus vector as a platform for gene therapy delivery. Nat Rev Drug Discov 18(5): 358-378.
18. Wu, S. C., Lo, Y. M., Lee, J. H., Chen, C. Y., Chen, T. W., Liu, H. W., Lian, W. N., Hua, K., Liao, C. C., Lin, W. J., et al. (2022). Stomatin modulates adipogenesis through the ERK pathway and regulates fatty acid uptake and lipid droplet growth. Nat Commun 13(1): 4174.