Weber Tom S, Dukes Mark, Miles Denise C, Glaser Stefan P, Naik Shalin H, Duffy Ken R
Hamilton Institute, Maynooth University, Maynooth, Ireland.
University of Strathclyde, Glasgow, UK.
BMC Syst Biol. 2016 Jun 30;10(1):43. doi: 10.1186/s12918-016-0290-3.
Cellular barcoding is a recently developed biotechnology tool that enables the familial identification of progeny of individual cells in vivo. In immunology, it has been used to track the burst-sizes of multiple distinct responding T cells over several adaptive immune responses. In the study of hematopoiesis, it revealed fate heterogeneity amongst phenotypically identical multipotent cells. Most existing approaches rely on ex vivo viral transduction of cells with barcodes followed by adoptive transfer into an animal, which works well for some systems, but precludes barcoding cells in their native environment such as those inside solid tissues.
With a view to overcoming this limitation, we propose a new design for a genetic barcoding construct based on the Cre Lox system that induces randomly created stable barcodes in cells in situ by exploiting inherent sequence distance constraints during site-specific recombination. We identify the cassette whose provably maximal code diversity is several orders of magnitude higher than what is attainable with previously considered Cre Lox barcoding approaches, exceeding the number of lymphocytes or hematopoietic progenitor cells in mice.
Its high diversity and in situ applicability, make the proposed Cre Lox based tagging system suitable for whole tissue or even whole animal barcoding. Moreover, it can be built using established technology.
细胞条形码技术是一种最近开发的生物技术工具，可在体内对单个细胞的后代进行谱系鉴定。在免疫学中，它已被用于追踪多个不同反应性T细胞在多种适应性免疫反应中的扩增规模。在造血研究中，它揭示了表型相同的多能细胞之间的命运异质性。大多数现有方法依赖于对细胞进行体外条形码病毒转导，然后将其过继转移到动物体内，这对某些系统有效，但排除了对天然环境中的细胞（如实体组织内的细胞）进行条形码标记。
为了克服这一限制，我们基于Cre Lox系统提出了一种新的基因条形码构建设计，该系统通过利用位点特异性重组过程中固有的序列距离限制，在细胞原位诱导随机产生的稳定条形码。我们鉴定出一种盒式结构，其可证明的最大编码多样性比以前考虑的Cre Lox条形码方法所能达到的多样性高几个数量级，超过了小鼠体内淋巴细胞或造血祖细胞的数量。
其高多样性和原位适用性使所提出的基于Cre Lox的标记系统适用于全组织甚至全动物条形码标记。此外，它可以使用现有技术构建。
