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The LeprCre and Glp1rCre mouse lines have been previously described21,44. LeprCre mice and the Glp1rCre lines were kindly provided by M. G. Myers and F. Reimann, respectively.

The POMCDre BAC construct was generated by inserting the sequence encoding Dre recombinase together with a kanamycin/neomycin resistance cassette from the pTEDre plasmid into the start codon of the POMC gene of RP11-124K7 BAC via Red/ET recombination. Primers containing POMC-specific homology arms, 5POMC-Dre: 5′-tccctccaatcttgtttgcctctgcagagactaggcctgacacgtggaaggccaccatgggtaagaagaaga-3′ and 3POMC Dre: 5′-accagctccacacatctatggaggtctgaagcaggagggccagcaacagggaggatttaatatttctgacgc-3′, were utilized for amplification. The ATG codon from POMC was replaced by that of the Dre recombinase. The BAC was linearized with PISceI in the presence of 2,5 mM spermidine and loaded onto a self-assembled CL-4b sepharose column (Sigma, CL4B200), equilibrated with injection buffer (5 mM Tris, 0.1 mM EDTA (pH 7.6)). The flow-through fraction with the highest concentration of digested BAC was chosen for pro-nucleus injection, performed by the team of R. Naumann at the MPI for Molecular Cell Biology and Genetics in Dresden. Cre and Dre transgenic animals were bred to C57BL/6N mice for maintenance in the mouse facility of the Max Planck Institute for Metabolism Research in Germany.

Generation of ROSA26lSlrSrZsGreen (ROSA26-CAGS-lox-STOP-lox-rox-STOP-rox-ZsGreen) mice has been described in a previous study20. These mice have been crossed to a ubiquitously expressed Deleter-Cre line to obtain R26rSrZsGreen mice20. ROSA26rSrlSltdTomato (ROSA26-CAGS-rox-STOP-rox-lox-STOP-lox-tdTomato-WPRE) mice were purchased from The Jackson Laboratory.

The knock-in lines ROSA26lSlrSrZsGreen (ROSA26-CAGS-lox-STOP-lox-rox-STOP-rox-ZsGreen), ROSA26lSlrSrhM3Dq (ROSA26-CAGS-lox-STOP-lox-rox-STOP-rox-hM3Dq-2A-ZsGreen-WPRE) and ROSA26lSlrSrEGFPL10a (ROSA26-CAGS-lox-STOP-lox-rox-STOP-rox-EGFPL10a-WPRE) were generated in-house. For generation of these mouse lines, a ROSA26 locus-targeting vector (B9-36) was designed in which both a loxP-flanked STOP cassette and a rox-flanked STOP cassette prevent CAGS promoter-driven expression of the corresponding functional transgenic construct. For hM3Dq, the 5′-primer (5AscRassle) overhang used for the amplification of the transgene contained an AscI site and a Kozak consensus sequence and the 3′-primer (3AscRassle) overhang contained an AscI site and one C to stay in frame for the 2A-ZsGreen translation. For the EGFPL10a, the 5′-primer overhang used for the amplification of the transgene contained an AscI site and a Kozak consensus sequence and the 3′-primer overhang contained an XmaI. The sequence-verified knock-in sequences of hM3Dq and EGFPL10a were cloned into the AscI-digested and AscI/XmaI-digested B9-36-targeting constructs, respectively. After vector transfection into Bruce 4 embryonic stem cells, they were subsequently screened for correct integration by standard Southern blot methods. To this end, a ROSA26 probe was used45 on EcoRI-digested clonal genomic DNA that indicated homologous recombination via detection of an additional 7.1-kb band besides the 16-kb endogenous ROSA26 band. To exclude random integration, a Neo probe was used that detected one single 7.1-kb band in case of a single correct ROSA26 insertion. Correctly targeted and verified embryonic stem cell clones were chosen for blastocyst injection carried out by Taconic Biosciences to obtain chimeric animals. Resulting chimeras were backcrossed with C57BL/6N mice to obtain germline transmission on a pure C57BL/6N background.

For the POMCDre ROSA26rSrZsGreen mouse line, the breeding scheme consisted of mating heterozygous POMCDre mice to homozygous ROSA26rx/rx mice of the ZsGreen construct. Resulting double transgenic Dre+/− ROSA26rx/wt mice were used as experimental animals and compared to C57BL/6N mice for metabolic phenotyping. Littermates of both sexes were used for experiments.

The mouse lines POMCDre LeprCre ROSA26lSlrSrZsGreen, POMCDre Glp1rCre ROSA26lSlrSrZsGreen, POMCDre LeprCre ROSA26rSrlSltdTomato, POMCDre Glp1rCre ROSA26rSrlSltdTomato, POMCDre LeprCre ROSA26lSlrSrhM3Dq, POMCDre Glp1rCre ROSA26lSlrSrhM3Dq, POMCDre LeprCre ROSA26lSlrSrEGFPL10a and POMCDre Glp1rCre ROSA26lSlrSrEGFPL10a were generated via mating heterozygous double transgenic mice to homozygous ROSA26fl;rx/fl;rx mice of the corresponding functional transgene construct (Extended Data Fig. Fig.2b).2b). Resulting triple transgenic Cre+/− Dre+/− ROSA26fl;rx/wt mice were used as experimental animals and compared to genotype controls as stated in the figure legends. Littermates of both sexes were used for experiments.

The C57BL/6N mouse line was purchased from Charles River. For metabolic phenotyping, both genders were used for experiments as indicated in the figure legends.

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