Generation of Brown Fat-Specific Knockout Mice Using a Combined Cre-LoxP, CRISPR-Cas9, and Adeno-Associated Virus Single-Guide RNA System

This protocol generates brown fat-specific knockout mice using a Cre-LoxP, CRISPR-Cas9, and adeno-associated virus single-guide RNA system. It includes a surgical technique to safely administer AAV into the brown fat. The main advantage of this technique is that it can rapidly generate tissue-specific knockouts without needing to breed mice selectively.

The approach to making tissue-specific knockout or knockdowns can be adapted to other tissues if the surgical procedure for injecting AAV is modified based on the target organ. To begin, administer the analgesic subcutaneously to the anesthetized UCP1 Cre Cas9 mouse to minimize and prevent postoperative pain and distress. Then shave the fur on its interscapular area using a shaver, and sterilize the surgical area with at least three alternating rounds of an iodine based or chlorhexidine based scrub, followed by 70%ethanol.

Using a scalpel, make a two centimeter incision in the skin above the brown adipose tissue between the scapulae to expose the interscapular fat. After making a single incision, cut the fat tissues on the border between the distal region of the fat tissues and muscles. Then using the surgical scissors in the dominant hand, peel the fat tissues off bluntly and vertically to expose the brown adipose tissue while pinching the fat tissues, including the brown adipose tissue, with the non-dominant hand.

Use Sulzer's vein as a landmark to indicate the precise brown adipose tissue location. Next, for each mouse, inject 20 microliters of adeno-associated virus or AAV slowly into each lobe of the two brown adipose tissues using a sharp 32 gauge needle connected to a 100 microliter Hamilton syringe. Confirm successful injection indicated by no leakages from the injected site during the AAV administration.

After ensuring homeostasis, place the exposed fat tissues back in their normal position to suture the edge of the fat tissues and muscle using a 5-0 absorbable monofilament thread. Finally, suture the open skin with 5-0 coated VICRYL undyed braided threads. The precise location of the brown adipose tissue cannot be identified without exposing it, however, excess exposure damage to the tissue.

The potential flaws that may have led to unsuccessful injections included injecting the AAV solution into the incorrect region, needle penetration through the tissue, and tissue ballooning due to an excessive volume of the AAV solution. The precise delivery of AAV single guide RNA to the brown adipose tissue was crucial to the protocol's success. The key to success while exposing the brown adipose tissue is surgical scissor technique, including the direction and depth of the scissors, and the location of Hamilton syringe.

This technique has a load-generating tissue-specific knockout animals to understand the molecular regulators of brown fat related energy metabolism.