Question

Question Set 3 (20 pts) As discussed in class, the pluripotential properties of hematopoietic stem cells (HSCs) were demonstrated by classic studies of marking cells by retroviral transduction and using these cells to reconstitute irradiated mice to make a "bone marrow chimera". Then one assesses fate based on integration sites in the haematopoietically derived cells. Recent studies such as those by Rodriguez-Fraticelli et al. 2018. Clonal Analysis of Lineage Fate in Native Hematopoiesis. Nature 553: 212 (PMC: 5884107) have used alternative methods including inducing transposon tagging to fate map hematopoietic cells. The system used in this paper is shown below. In general, it is a mouse transgenic model where, when treated with Doxycycline, transposition is induced and cells will be differentially "barcoded" based on integration sites. Marking cells with a transposable element. Key features 1) Engineered transgenic mice so mice have inducible transposon system in their genome, you know the sequences and can follow transposition through PCR and sequencing, including single cell sequencing; 2) Inducible: adding Dox induces transposition; 3) Transposition should be random and unbiased as illustrated in (b). b Unlabelled cells Labelled cells Labelled progeny Rosa26 M2-TA Dox collate0 39 - Rosa26 M2-ATA collat - - collat To COS Random locus coltal ਟੈਕGose ee CAGOS Ds Red Random locus Unique integration site 3a. (7 pts) What is a difference between using this approach versus the classic approach of using retroviruses to tag cells and what kind of added information might this experimental system provide? 3b. (13 pts) Data from this paper and others using similar strategies to examine the contribution of HSC to adult hematopoiesis have suggested, at least in adult animals, that the hematopoietic stem cell population may not contribute equally to all lineages, and there may be a bias towards certain lineages such as megakarocyte progenitors and related lineages. Propose a model that would allow for the maintenance of other myeloid and lymphoid lineages if HSCs were not directly contributing to their development in steady state hematopoiesis.

Answer 1

3.a. By the use of transposons, we understand the dynamics of lineage coupling whereas Retroviral vectors are vehicles for gene transfer. However, difficult to achieve long-term expression mediated by integrated proviruses in primary cells due to Retroviral regulatory elements that are repressed in hematopoietic stem cells.