Animals
Mice were group housed in standard housing under a 12–12 h light–dark cycle with ad libitum access to chow diet and water, with the room temperature kept around 22 °C and humidity kept between 30–80% (not controlled). Mice were single housed for food intake measurement and held at 30 °C for thermoneutral exposure experiments. Mice (aged at least 6 weeks) from the following strains were used for this study: wild-type (WT) C57BL/6J (Jackson stock, 000664), B6.Cg-Gt(ROSA)26Sortm9(CAG-tdTomato)Hze/J (Jackson, 007909, Ai9), Pirt-Cre51, Scn10a-Cre52. Both genders were used for anatomical mapping studies, and male mice were used for in vivo functional experiments. All of the experimental protocols were approved by The Scripps Research Institute Institutional Animal Care and Use Committee and were in accordance with the guidelines from the NIH.
Viruses
CAV2 was obtained from CNRS Vector Core. rAAVretro-Cre was obtained from Boston Children’s Hospital and Janelia. PHP.S-Cre, PHP.S-DIO-sfGFP (gift from D. Gibbs) was obtained from Janelia. PHP.S-TdTomato (Addgene, 59462), PHP.S-mScarlet (gift from the Deisseroth laboratory and cloned in-house), ROOT-Cre (Addgene, 51904), ROOT-EYFP (cloned in-house), PHP.S-mCherry-flex-DTA (Addgene, 58536), ROOT-mScarlet, AAV9-mScarlet and PHP.S-mScarlet were packaged in-house using published protocol53. AAVs produced in-house were titrated by qPCR before aliquoting into 6–10 μl and flash-frozen for long-term storage.
In vivo selection of ROOT
Plasmids
The plasmids used for in vivo selection were adapted from the previous publication54. rAAV-pUBC-sfGFP-Cap and AAV2/9-REP-AAP was generated from pUBC-mCherry-rAB (Addgene, 115239), pUCmini-iCAP-PHP.S (Addgene, 103006), pAAV2/8 (Addgene, 112864). No in-cis-Lox module or transgenic Cre lines were used given the anatomical separation of DRGs and iWAT. The ROOT capsid library was generated by inserting random heptamers using NNK degenerate primers (Integrated DNA technologies) between the 588 and 589 sites of AAV9 by Gibson assembly as previously described54.
AAV capsid library production
The viral libraries were produced as previously described53,54. In brief, only 10 ng of rAAV-pUBC-sfGFP-Cap library DNA was transfected in HEK293FT (Invitrogen R70007) cells per 150 mm plate, and the virus was collected after 60 h for purification.
DNA recovery and sequencing
The resulting AAV capsid library was injected bilaterally into the iWAT of C57Bl/6J male mice at 109 viral genomes (VGs) per fat pad. rAAV genomes were recovered from T12–L3 DRGs from injected mice two weeks after injection using the DNeasy Blood and Tissue kit (Qiagen). The recovered viral DNA was amplified for two rounds against a fragment containing the heptamer insertion with Q5 high-fidelity polymerase (New England Biolabs). The amplified products were cleaned up and processed for complete amplicon sequencing at Massachusetts General Hospital.
NGS data alignment and processing
Raw FASTQ files from NGS runs were aligned to an AAV9-template DNA fragment containing the 21 bp diversified region between amino acids 588 and 589 using SAMtools (v.1.10). The abundance of each 21 bp sequence in all of the recovered sequences with the heptamer insertion was quantified.
Surgery
Mice were anaesthetized with isoflurane (4% for induction and 1.5–2% for maintenance), with their skin at the surgical area shaved and hair removed and sterilized using ethanol and iodine. After surgery, the mice were given a subcutaneous injection of flunixin and topical antibiotic ointment for post-operative care.
Retrograde tracer labelling of sensory neurons
For injection into the iWAT, a lateral incision was made on the flank skin of each side. For injection into the eWAT, a lateral incision was made on the lower abdominal wall. For injection into the iBAT, a midline incision was made in the interscapular region. For injection into the skin, an intradermal injection was performed. A Hamilton…
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