Direct lentivirus injection for fast and efficient gene transfer into brown and beige adipose tissue

Authors

  • Aileen Balkow Institute of Pharmacology and Toxicology, University Hospital Bonn, University of Bonn, Germany
  • Linda S. Hoffmann Institute of Pharmacology and Toxicology, University Hospital Bonn, University of Bonn, Germany
  • Katarina Klepac Institute of Pharmacology and Toxicology, University Hospital Bonn, University of Bonn, Germany; Research Training Group 1873, University of Bonn, Germany
  • Anja Glöde Institute of Pharmacology and Toxicology, University Hospital Bonn, University of Bonn, Germany; BIGS DrugS International Graduate School, University of Bonn, Germany
  • Thorsten Gnad Institute of Pharmacology and Toxicology, University Hospital Bonn, University of Bonn, Germany
  • Katrin Zimmermann Institute of Pharmacology and Toxicology, University Hospital Bonn, University of Bonn, Germany
  • Alexander Pfeifer Institute of Pharmacology and Toxicology, University Hospital Bonn, University of Bonn, Germany; Research Training Group 1873, University of Bonn, Germany; BIGS DrugS International Graduate School, University of Bonn, Germany; PharmaCenter, University of Bonn, Germany

DOI:

https://doi.org/10.14440/jbm.2016.123

Keywords:

beige adipose tissue, brown adipose tissue, gene transfer, lentivirus

Abstract

Brown adipose tissue is a special type of fat contributing to energy expenditure in human newborns and adults. Moreover, subcutaneous white adipose tissue has a high capacity to adapt an energy-consuming, brown-like/beige phenotype. Here, we developed an easy to handle and fast to accomplish method to efficiently transfer genes into brown and beige fat pads in vivo. Lentiviral vectors are directly injected into the target fat pad of anaesthetized mice through a small incision using a modified, small needle connected to a microsyringe, which is well suited for infiltration of adipose tissues. Expression of the target gene can be detected in brown/beige fat one week after injection. The method can be applied within minutes to efficiently deliver transgenes into subcutaneous adipose tissues. Thus, this protocol allows for studying genes of interest in a timely manner in murine brown/beige fat and could potentially lead to new gene therapies for obesity. Brown adipose tissue is a special type of fat contributing to energy expenditure in human newborns and adults. Moreover, subcutaneous white adipose tissue has a high capacity to adapt an energy-consuming, brown-like/beige phenotype. Here, we developed an easy to handle and fast to accomplish method to efficiently transfer genes into brown and beige fat pads in vivo. Lentiviral vectors are directly injected into the target fat pad of anaesthetized mice through a small incision using a modified, small needle connected to a microsyringe, which is well suited for infiltration of adipose tissues. Expression of the target gene can be detected in brown/beige fat one week after injection. The method can be applied within minutes to efficiently deliver transgenes into subcutaneous adipose tissues. Thus, this protocol allows for studying genes of interest in a timely manner in murine brown/beige fat and could potentially lead to new gene therapies for obesity.

References

Ng M, Fleming T, Robinson M, Thomson B, Graetz N, et al. (2014) Global, regional, and national prevalence of overweight and obesity in children and adults during 1980-2013: a systematic analysis for the Global Burden of Disease Study 2013. Lancet 384: 766-781.

Pfeifer A, Hoffmann LS (2014) Brown, beige, and white: the new color code of fat and its pharmacological implications. Annu Rev Pharmacol Toxicol 55: 207-227.

Cannon B, Nedergaard J (2004) Brown adipose tissue: function and physiological significance. Physiol Rev 84: 277-359.

Klingenspor M, Herzig S, Pfeifer A (2013) Brown fat develops a brite future. Obes Facts 5: 890-896.

van Marken Lichtenbelt WD, Vanhommerig JW, Smulders NM, Drossaerts JM, Kemerink GJ, et al. (2009) Cold-activated brown adipose tissue in healthy men. N Engl J Med 360: 1500-1508.

Cypess AM, Lehman S, Williams G, Tal I, Rodman D, et al. (2009) Identification and importance of brown adipose tissue in adult humans. N Engl J Med 360: 1509-1517.

Virtanen KA, Lidell ME, Orava J, Heglind M, Westergren R, et al. (2009) Functional brown adipose tissue in healthy adults. N Engl J Med 360: 1518-1525.

Yoneshiro T, Aita S, Matsushita M, Kayahara T, Kameya T, et al. (2013) Recruited brown adipose tissue as an antiobesity agent in humans. J Clin Invest 123: 3404-3408.

van der Lans AA, Hoeks J, Brans B, Vijgen GH, Visser MG, et al. (2013) Cold acclimation recruits human brown fat and increases nonshivering thermogenesis. J Clin Invest 123: 3395-3403.

Harms M, Seale P (2013) Brown and beige fat: development, function and therapeutic potential. Nat Med 19: 1252-1263.

Rosen ED, Spiegelman BM (2014) What we talk about when we talk about fat. Cell 156: 20-44.

Seale P, Conroe HM, Estall J, Kajimura S, Frontini A, et al. (2010) Prdm16 determines the thermogenic program of subcutaneous white adipose tissue in mice. J Clin Invest 121: 96-105.

Schulz TJ, Huang TL, Tran TT, Zhang H, Townsend KL, et al. (2010) Identification of inducible brown adipocyte progenitors residing in skeletal muscle and white fat. Proc Natl Acad Sci U S A 108: 143-148.

Hoffmann LS, Larson CJ, Pfeifer A (2015) cGMP and Brown Adipose Tissue. Handb Exp Pharmacol.

Pfeifer A, Verma IM (2001) Gene therapy: promises and problems. Annu Rev Genomics Hum Genet 2: 177-211.

Haas B, Mayer P, Jennissen K, Scholz D, Berriel Diaz M, et al. (2009) Protein kinase G controls brown fat cell differentiation and mitochondrial biogenesis. Sci Signal 2: ra78.

Mitschke MM, Hoffmann LS, Gnad T, Scholz D, Kruithoff K, et al. (2013) Increased cGMP promotes healthy expansion and browning of white adipose tissue. FASEB J 27: 1621-1630.

Fujiwara K, Hasegawa K, Ohkumo T, Miyoshi H, Tseng YH, et al. (2012) Necdin controls proliferation of white adipocyte progenitor cells. PLoS One 7: e30948.

Gnad T, Scheibler S, von Kugelgen I, Scheele C, Kilic A, et al. (2014) Adenosine activates brown adipose tissue and recruits beige adipocytes via A2A receptors. Nature 516: 395-399.

Pfeifer A, Lehmann H Pharmacological potential of RNAi--focus on miRNA. Pharmacol Ther 126: 217-227.

Pfeifer A, Hofmann A (2009) Lentiviral transgenesis. Methods Mol Biol 530: 391-405.

Kang S, Kong X, Rosen ED (2014) Adipocyte-specific transgenic and knockout models. Methods Enzymol 537: 1-16.

Liu X, Magee D, Wang C, McMurphy T, Slater A, et al. (2014) Adipose tissue insulin receptor knockdown via a new primate-derived hybrid recombinant AAV serotype. Mol Ther Methods Clin Dev 1.

Verma IM, Somia N (1997) Gene therapy -- promises, problems and prospects. Nature 389: 239-242.

Daya S, Berns KI (2008) Gene therapy using adeno-associated virus vectors. Clin Microbiol Rev 21: 583-593.

Yang L, Bailey L, Baltimore D, Wang P (2006) Targeting lentiviral vectors to specific cell types in vivo. Proc Natl Acad Sci U S A 103: 11479-11484.

Ussar S, Lee KY, Dankel SN, Boucher J, Haering MF, et al. (2014) ASC-1, PAT2, and P2RX5 are cell surface markers for white, beige, and brown adipocytes. Sci Transl Med 6: 247ra103.

Pauwels K, Gijsbers R, Toelen J, Schambach A, Willard-Gallo K, et al. (2009) State-of-the-art lentiviral vectors for research use: risk assessment and biosafety recommendations. Curr Gene Ther 9: 459-474.

Trueck C, Zimmermann K, Mykhaylyk O, Anton M, Vosen S, et al. (2012) Optimization of magnetic nanoparticle-assisted lentiviral gene transfer. Pharm Res 29: 1255-1269.

Jennissen K, Haas B, Mitschke MM, Siegel F, Pfeifer A (2013) Analysis of cGMP signaling in adipocytes. Methods Mol Biol 1020: 175-192.

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Published

2016-07-16

How to Cite

1.
Balkow A, Hoffmann LS, Klepac K, Glöde A, Gnad T, Zimmermann K, Pfeifer A. Direct lentivirus injection for fast and efficient gene transfer into brown and beige adipose tissue. J Biol Methods [Internet]. 2016Jul.16 [cited 2021May8];3(3):e48. Available from: https://jbmethods.org/jbm/article/view/123

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Section

Protocols