Univ Paris Est Creteil, INSERM, IMRB, F-94010, Creteil, France.
Ecole nationale vétérinaire d'Alfort, IMRB, F-94700, Maisons-Alfort, France.
Stem Cell Res Ther. 2023 Oct 13;14(1):294. doi: 10.1186/s13287-023-03506-1.
Ever since its introduction as a genetic tool, the Cre-lox system has been widely used for molecular genetic studies in vivo in the context of health and disease, as it allows time- and cell-specific gene modifications. However, insertion of the Cre-recombinase cassette in the gene of interest can alter transcription, protein expression, or function, either directly, by modifying the landscape of the locus, or indirectly, due to the lack of genetic compensation or by indirect impairment of the non-targeted allele. This is sometimes the case when Cre-lox is used for muscle stem cell studies. Muscle stem cells are required for skeletal muscle growth, regeneration and to delay muscle disease progression, hence providing an attractive model for stem cell research. Since the transcription factor Pax7 is specifically expressed in all muscle stem cells, tamoxifen-inducible Cre cassettes (CreERT2) have been inserted into this locus by different groups to allow targeted gene recombination. Here we compare the two Pax7-CreERT2 mouse lines that are mainly used to evaluate muscle regeneration and development of pathological features upon deletion of specific factors or pathways. We applied diverse commonly used tamoxifen schemes of CreERT2 activation, and we analyzed muscle repair after cardiotoxin-induced injury. We show that consistently the Pax7-CreERT2 allele targeted into the Pax7 coding sequence (knock-in/knock-out allele) produces an inherent defect in regeneration, manifested as delayed post-injury repair and reduction in muscle stem cell numbers. In genetic ablation studies lacking proper controls, this inherent defect could be misinterpreted as being provoked by the deletion of the factor of interest. Instead, using an alternative Pax7-CreERT2 allele that maintains bi-allelic Pax7 expression or including appropriate controls can prevent misinterpretation of experimental data. The findings presented here can guide researchers establish appropriate experimental design for muscle stem cell genetic studies.
自从 Cre-lox 系统作为一种遗传工具被引入以来，它已经被广泛应用于健康和疾病背景下的体内分子遗传学研究，因为它允许时间和细胞特异性的基因修饰。然而，Cre 重组酶盒的插入会改变目的基因的转录、蛋白表达或功能，这可能是直接的，通过改变基因座的景观，也可能是间接的，由于缺乏遗传补偿或通过间接损害非靶向等位基因。在使用 Cre-lox 进行肌肉干细胞研究时，就会出现这种情况。肌肉干细胞是骨骼肌生长、再生和延缓肌肉疾病进展所必需的，因此为干细胞研究提供了一个有吸引力的模型。由于转录因子 Pax7 特异性地表达在所有的肌肉干细胞中，不同的研究小组已经将诱导型 Cre 盒（CreERT2）插入到这个基因座中，以允许靶向基因重组。在这里，我们比较了两种主要用于评估特定因素或通路缺失后肌肉再生和病理特征发展的 Pax7-CreERT2 小鼠品系。我们应用了多种常用的 CreERT2 激活方案，分析了心脏毒素诱导损伤后的肌肉修复。结果表明，Pax7-CreERT2 等位基因在 Pax7 编码序列中靶向（敲入/敲除等位基因）会导致再生的固有缺陷，表现为损伤后修复延迟和肌肉干细胞数量减少。在缺乏适当对照的遗传消融研究中，这种固有缺陷可能被错误地解释为所关注因素的缺失所引起的。相反，使用另一种维持双等位基因 Pax7 表达的 Pax7-CreERT2 等位基因或包括适当对照可以防止对实验数据的错误解释。这里提出的发现可以指导研究人员为肌肉干细胞遗传研究建立适当的实验设计。
