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Table 1 Strategies for effective gene recombination in adult satellite cells and their progeny during muscle repair

From: Pax7 is back

TMX delivery

Recovery period

Gene deletion analysis

Escaper cell analysis

TMX injection at 3 mg/day for 5 consecutive daysa

Allow >15 days of TMX chase prior to muscle injury to target quiescent SCs and minimize lingering TMX activity

Isolate purified SCs by FACS, or from single fibers from each experimental animalb

Measure gene or protein levels over timec

Five daily injections prior to injury followed by continuous TMX feeding delivered in chow (1 mg TMX per day)

Pros: Temporal identity of cell type and state

Measurement of protein by western blot or immunohistochemistry

In vitro: Measure gene/protein immediately after isolation and after approximately 10 days in culture

Minimize recombination of transient cell populations

Quantify DNA directly at the gene locus using site-specific primers flanking the loxP sites

In vivo: Measure gene/protein from SCs isolated from uninjured and regenerated muscle (>30 days after injury)

Cons: Escaper cell contribution

No recovery period

Pros: Maximize targeting of both stable and transient cell populations

Minimize escaper cell contribution

Cons: Lose cell type and state resolution

  1. aRecommendation based on 30-gram adult mouse.
  2. bDue to variable recombination efficiency between mice, assessment of recombination in every mouse is strongly advised. In addition, I caution the use of R26R-based fluorescent reporters as a surrogate to assess recombination of a gene of interest.
  3. cIn the presence of escaper cells, gene/protein levels of the targeted allele will increase over time if the recombined gene has a loss-of-function phenotype. If recombination results in a gain-of-function mutant or acts redundantly, then gene/protein levels will decrease or not change, respectively. In the absence of escaper cells, the gene/protein of interest will not be detectable.