Development of an In Vivo Model to Study Clonal Lineage Relationships in Hematopoietic Cells Using Brainbow2.1/Confetti Mice

Abstract

Hematopoietic stem cells maintain the homeostasis of all blood cell progeny during development and repopulation-demanding events. To study the lineage relationships during hematopoiesis, increasingly complex cell tracing models are being developed. In this study, we describe adaptations to the original R26R-Confetti mouse model, which subsequently offers a relatively easy approach to study low complexity clonality during hematopoiesis, with special focus on B and T lymphocyte development. This protocol employs spatiotemporal Cre expression controlled by gammaretroviral transduction for efficient fluorescent protein cell marking. Transplantation of fluorescently marked Lin- cKit+ hematopoietic progenitor cells into Rag1-/- mice, resulted in the visualization of differentially contributing stem cell clones to various lineages. Our methodology is useful to study questions in fundamental and preclinical hematopoietic research and in vivo B- and T-cell development.

Overexpression of Mothers Against Decapentaplegic Homolog 7 Activates the Yes‐Associated Protein/NOTCH Cascade and Promotes Liver Carcinogenesis in Mice and Humans

“…Next, we applied a stochastic multicolor Cre-reporter R26R-confetti homozygous mice (will be referred as "Confetti mice") for lineage tracing. (39,40) Hepatocytespecific adeno-associated virus 8 (AAV8)-thyroxine binding globulin (TBG)-Cre (41) was injected into 4-week-old Confetti mice to trigger the expression of fluorescent proteins. A plasmids mixture of c-Myc/ MCL1/Smad7/SB was delivered into mouse liver 2 weeks later (Supporting Fig.…”

Fluorescence Microscopy—An Outline of Hardware, Biological Handling, and Fluorophore Considerations

“…The “Brainbow” strategy relies on a handful of spectroscopically distinct FPs to provide information of different cellular types and environments, by emitting a broad palette of detectable hues [ 183 ]. Hematopoietic stem cells, which give rise to B and T cells, have recently been imaged by transplanting a range of fluorescently labelled cells with differing emission profiles, to provide information on hematopoietic cell lineage in Rag1 −/− mice using confocal microscopy [ 184 ]. Similarly, live pluripotent stem cells were imaged by confocal microscopy to track their eventual derivatisation using the Brainbow technique ( Figure 2 ) [ 185 ].…”

Intravital microscopy imaging of kidney injury and regeneration

“…Taking advantage of the widely used Cre/Loxp system, the researchers designed a genetic strategy called "Brainbow" [26,27]. This Brainbow transgenic mouse can randomly express a variety of fluorescent proteins (XFPs) under Cre-mediated recombination [27,28]. The first generation of brainbow mice (Braindow-1) used Cremediated excision of fragments between Loxp sites to induce recombination events [27].…”

“…The first generation of brainbow mice (Braindow-1) used Cremediated excision of fragments between Loxp sites to induce recombination events [27]. In Brainbow-2, Cre reverses the DNA fragments defined by the LoxP site and connects them in opposite directions to produce multiple recombination results [27,28]. The R26R-Confetti mice were created to study the fate map of intestinal stem cells [9].…”