Institute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, Stanford, CA, 94305, USA.
Experimental Animal Division, Bioscience Education and Research Support Center, Akita University, Akita, 010-8543, Japan.
Sci Rep. 2022 Jun 17;12(1):10223. doi: 10.1038/s41598-022-14467-5.
Animal chimeras are widely used for biomedical discoveries, from developmental biology to cancer research. However, the accurate quantitation of mixed cell types in chimeric and mosaic tissues is complicated by sample preparation bias, transgenic silencing, phenotypic similarity, and low-throughput analytical pipelines. Here, we have developed and characterized a droplet digital PCR single-nucleotide discrimination assay to detect chimerism among common albino and non-albino mouse strains. In addition, we validated that this assay is compatible with crude lysate from all solid organs, drastically streamlining sample preparation. This chimerism detection assay has many additional advantages over existing methods including its robust nature, minimal technical bias, and ability to report the total number of cells in a prepared sample. Moreover, the concepts discussed here are readily adapted to other genomic loci to accurately measure mixed cell populations in any tissue.
动物嵌合体被广泛用于生物医学发现，从发育生物学到癌症研究。然而，在嵌合体和镶嵌组织中准确量化混合细胞类型受到样本制备偏差、转基因沉默、表型相似性和低通量分析管道的影响。在这里，我们开发并表征了一种液滴数字 PCR 单核苷酸区分检测方法，以检测常见白化和非白化小鼠品系之间的嵌合性。此外，我们验证了该检测方法与所有固体器官的粗裂解物兼容，极大地简化了样本制备。与现有方法相比，这种嵌合检测方法具有许多额外的优势，包括其稳健性、最小的技术偏差以及报告准备样品中总细胞数的能力。此外，这里讨论的概念可以很容易地应用于其他基因组座，以准确测量任何组织中的混合细胞群。
