Scientific Papers

Pax7 haploinsufficiency impairs muscle stem cell function in Cre-recombinase mice and underscores the importance of appropriate controls | Stem Cell Research & Therapy


Study analysis

In June 2022 we mined PubMed for all papers that cited one or more of the four publications that describe the Pax7CreERT2(Fan) allele by the group that generated it [1, 7, 9, 10]. We then removed reviews and duplicates. The remaining papers were categorized as shown in Table 1 and as summarized in Fig. 1A.

Table 1 Publications citing one or more of the four publications that describe the Pax7CreERT2(Fan) allele by the group that generated it

Mice

The following mouse (Mus musculus) lines were provided by the corresponding laboratories as described: Pax7CreERT2(Fan) [7], Pax7+LacZ [12], Pax7CreERT2(Gaka) (G. Kardon; Jackson Laboratories, stock 017763; [3]. Animals were handled as per French and European Union guidelines, and protocols were approved by the French Ministry of Higher Education, Research and Innovation following favorable opinion of the ethics committee 16/Anses-ENVA-UPEC (Project No: 20–027 #27431). The same ethics committee approved our study design in terms of animal numbers taking into account statistical power and the “Reduction” aspect of the 3R principle. We analyzed a minimum of three animals per genotype per line. Adult (8–12 week old) animals were imported weekly from the breeding facility at Orleans (France) to the experimental facility at Creteil (France) and they were given minimum seven days to adjust to the new conditions. A priori exclusion criteria were (i) death before inclusion to an experimental group (details in Table 2) and ii) the rare event that the TA was not fully injected, or it broke before cryosectioning and analysis (details in Table 2). In the above cases, a new control-mutant set was imported into the experimental facility; thus, the compared groups occasionally have n > 3. A total of 76 animals were analyzed in this study.

Table 2 List of animals excluded from analysis

In both facilities mice are kept in rooms, cages, and enrichment environments that are up to the highest EU and French standards and regulations; both facilities have been audited and certified and further information on housing and husbandry can be requested to the facility heads. In the context of the tamoxifen and injury experimental procedures of this study (details below), the following methods were established and approved by the ethics committee to minimize or deplete pain and suffering: (i) mouse handling only by authorized researchers that have passed the two-week initial animal experimentation training and relevant continuous education required in France, (ii) mouse follow-up by experienced animal caretakers, including weekly checks; as a general practice, when signs of suffering are presented (e.g., distress, muscular atrophy, cachexia, respiratory difficulties, ulcers, etc.), the animals are euthanized, (iii) placement of hydrogel and wet food on cage floor, in case of reduced mobility after the muscle injury procedure [in that case the animal is checked more often for the signs listed in (ii)], (iv) acclimatization of at least seven days after importation to the experimental facility, (v) animal caretaker training for housing amelioration, for example cage enrichment with cardboard tunnels or plastic shelters.

Tamoxifen treatment and injury

Adult (8–12 weeks of age) male mice were placed on tamoxifen diet (Envigo; TD.55125.I) for two (Fig. 1G-J) or five weeks (Fig. 2), as indicated in the figure, and were observed regularly for signs of poor tolerance (weight loss, suffering, apathy, dehydration, etc.). For muscle injuries, mice were anesthetized with 3% isoflurane, and injured by intramuscular injection of 50 uL cardiotoxin at 10 umol (Latoxan; L8102). Specifically, in a dedicated room of the experimental animal facility, mice were put in dedicated transparent plastic cages that had isoflurane and oxygen flow until anesthetized; once anesthetized then they were transferred on warming plates to be injected under continuing inhalation anesthesia. Once injected they were transferred back to their cage and observed until waking up, followed by daily observation for signs of poor tolerance (weight loss, suffering, apathy, dehydration, etc.). At seven (Figs. 1, 2) or 28 days post-injury (Fig. 2), mice were sacrificed by cervical dislocation and their TA muscles were collected for analysis. We observed no adverse events that could be attributed to the procedures in which the animals were subjected.

Immunostaining

Muscles were frozen fresh in liquid nitrogen-cooled isopentane and sectioned at 8um with a Leica cryostat. Sections were first fixed with 4% paraformaldehyde for 10 min and washed 3 times with PBS. The sections were then permeabilized with 0.5% Triton-X (Sigma) for 8 min, washed with PBS, then blocked with 5% BSA (Jackson Laboratories 001–000-162) for 1 h at room temperature. Sections were then incubated with the primary antibodies overnight at 4 oC. The next day, samples were washed with PBS, incubated with the secondary antibodies for 1 h at room temperature, washed with PBS, and mounted.

For PAX7 staining, samples were permeabilized with cold methanol for 6 min instead of Triton-X, washed with PBS, then boiled in citric acid (10 mM, pH6) for 3 min, then let cool down at RT for 30 min. Sections were then washed with PBS, blocked with 5% BSA and incubated with Fab (Interchim; 115–007-003) for 30 min at RT. The staining was then completed as described above.

All antibodies are listed in Table 3.

Table 3 List of antibodies

Histology

Muscles were frozen fresh in liquid nitrogen-cooled isopentane and sectioned at 8 mm with a Leica cryostat. Sections were fixed with 4% paraformaldehyde for 20 min at room temperature and washed twice with PBS (10 min per wash). Nuclei were stained with hematoxylin (Sigma) for 25 min and cytoplasms were counterstained with eosin (Sigma) for 40 s. The sections were then dehydrated with 30 s passages through increasing concentrations of ethanol (30%, 50%, 70%, 85%, 95%), followed by 15 min in absolute ethanol.

Statistical analyses

For comparison between two groups, unpaired Student’s t test was performed to calculate p values and to determine statistically significant differences using Graphpad’s Prism software (*p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001). The experimental unit was single animals (data per animal are presented in Figs. 1, 2; exact values can be provided upon request). We primarily compared the control and mutant groups of each line (Pax7CreERT2(Fan): Ctrl VS CreERT2, Pax7CreERT2(Gaka): Ctrl VS CreERT2, Pax7LacZ: Ctrl VS het). When indicated in the figure, we also compared mutants of different lines (Fan-CreERT2 VS Gaka-CreERT2 or Fan-CreERT2 vs LacZ-het).



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