This protocol presents the SPH CRISPR Activation System as an alternative strategy to conventional viral vectors to perform gain function assays in adipocytes. It allows the overexpression of single or multiple adipocyte endogenous genes within the complex cellular environment of adipose tissue by delivering a customized single guide RNA using an adeno associated virus. The challenging steps of this protocol are related to cloning the single guide RNA and the production and injection of adeno-associated virus into the adipose tissue.
To begin, design single guide RNA or sgRNA for CRISPR activation using chopchop or any other suitable tool. Design the sgRNA targeting the PRDM16 gene using the following parameters:Target as PRDM16 in a Mus musculus using a CRISPR/Cas9 and for as activation. Add overhangs to the sgRNA to match the Sac1 restriction site in the vector backbone PAAVU6GRNACBHM cherry.
Include 5'AGCT 3'where n corresponds to nucleotides. Obtain 3'sgRNA reverse complement sequence using the indicated tool. Next, for annealing of single-stranded complimentary oligonucleotides, add one microliter of each 5'and 3'single-stranded oligonucleotide, one microliter of T4 ligase buffer, 0.5 microliters of T4 polynucleotide kinase, and 6.5 microliters of water for a final reaction volume of 10 microliters.
Anneal the complimentary single-stranded oligonucleotides using a thermocycler at 37 degrees Celsius for 30 minutes and 95 degrees Celsius for five minutes, followed by a ramp-down rate of five degrees Celsius per minute. Then for ligation of annealed sgRNA oligonucleotides add 25 nanograms of plasmid PAAVU6GRNACBHM cherry to two microliters of annealed sgRNA Oligonucleotides, one microliters of Sac1 enzyme, two microliters of 10X T4 DNA ligase buffer, one microliter of T4 DNA ligase and water to a final reaction volume of 10 microliters. Using a thermocycler, perform ligation by incubating the reaction mixture for 15 cycles at 37 degrees Celsius for five minutes and 25 degrees Celsius for five minutes followed by holding at four degrees Celsius.
Next, transform the competent Escherichia coli DH10B cells with four microliters of the ligation product by heat shock at 42 degrees Celsius for 45 seconds and spread on an agar plate containing 10 micrograms per milliliter Ampicillin. Confirm transformed colonies by Colony Polymerase Chain Reaction or PCR using the PCR Master Mix, pick the colony and mix it with five microliters of Master Mix, 0.1 microliters of universal primer, 0.1 microliter of sgRNA reverse primer and five microliters of water. Run the PCR using initial denaturation at 94 degrees Celsius for two minutes, followed by 35 cycles of denaturation at 94 degrees Celsius for 20 seconds, annealing for 30 seconds at 60 degrees Celsius, extension at 72 degrees Celsius for 30 seconds, and a final elongation step at 72 degrees Celsius for five minutes.
After PCR, resolve the DNA using agarose gel electrophoresis in 0.5X TAE Buffer at 90 volts for 30 minutes. Submit positive samples for Sanger Sequencing using universal primer. Next, purify the plasmid from a positive clone using a plasmid purification kit following the manufacturer's instructions.
Place the anesthetized mouse in the supine position and shave a small area on the flanks proximal to the hip joints for inguinal white adipose tissue or IWAT injections with a shaver. Add depilatory cream for five minutes. Remove residual cream with water to avoid skin burns.
Disinfect the skin using three alternating rounds of applying the povidone iodine solution on the skin with clean gauze and 70%alcohol. Then, make a one to two centimeter incision with sterilized scissors in the proximal area of the joints and hold the skin open using forceps to expose the fat depot. The WAT can be attached to the skin on both sides extending it from the beginning from the back and towards the testes.
Using forceps, gently pull the fat depot upward through the incision to ensure the injection is at the correct location and depth. Fill the microliter syringe with 2.5 microliters of the adeno-associated virus or a AAV containing the sgRNA targeting the endogenous PRDM16 gene. Carefully insert the needle at a 30-45 degree angle into the IWAT.
Repeat the injection five times into different locations of the tissue to homogeneously infect the whole IWAT fat pad. Place the mouse on the heating pad until consciousness is regained. Monitor the animal every 10 to 15 minutes until it fully recovers.
After the animal regains consciousness observe the locomotive profiling, which should be linear and have no signs of distress or pain. See the stromal vascular cells or SVF's derived from adipo SPH mouse IWAT into a 6-Well plate containing complete DMEM medium for one to two hours, aspirate the medium, wash the well twice using PBS, and replace it with fresh, complete medium. Incubate the cells at 37 degrees Celsius, 5%carbon dioxide and 95%humidity until the cells reach 70-80%confluency.
At Day 0, induce differentiation by treating the cells with the induction medium and after two days replace the induction medium with the maintenance medium. Again after two days, at day four, replace the maintenance medium with a fresh maintenance medium for 2-3 days. Change the maintenance medium every 48 hours until the preadipocytes are fully differentiated into adipocytes.
Observe the mature adipocytes using light microscopy as the differentiated cells appear to be loaded with lipid droplets. After growing the cells on a 6-Well culture plate with the complete medium until the cells reach 70-80%confluency, mix 5.6*10^10 viral genome per microliter of AAV-carrying sgRNA PRDM16 with two milliliters of complete medium and hexadimethrine bromide. Transduce the cells by replacing the complete medium and adding the complete medium containing AAV.
Incubate the transduced cells for 12 hours at 37 degrees Celsius 95%humidity and 5%carbon dioxide. Split and see the cells as described for cell proliferation and differentiation into beige adipocytes. The immunofluorescence images from IWAT of adipo SPH mice demonstrated the expression of dCas9 in both the control and PRDM16 groups.
SPH-induced PRDM16 expression clearly induced a widespread accumulation of multi ocular beige adipocytes into IWAT. Moreover, quantitative PCR revealed an increased expression of PRDM16 and the thermogenic gene program in the PRDM16 group compared to the control. Similarly, in vitro gene expression analysis confirmed the increased expression of the endogenous PRDM16 gene and thermogenic genes in the PRDM16 overexpression group compared to the control.
The SPH-induced PRDM16 expression resulted in higher basal and maximal oxygen consumption than that in control cells. Importantly, the data indicated enhanced uncoupled respiration in the PRDM16 group compared with that in the control group. Adipo SPH model is suitable for investigating multiple genes and regulatory elements within adipocytes.
This method opens up the possibility of a deeper understanding of beige fat biology.
