Gene transfer to pre-hematopoietic and committed hematopoietic precursors in the early mouse Yolk Sac: a comparative study between in situ electroporation and retroviral transduction

Abstract

<p>Abstract</p> <p>Background</p> <p>Hematopoietic development in vertebrate embryos results from the sequential contribution of two pools of precursors independently generated. While intra-embryonic precursors harbour the features of hematopoietic stem cells (HSC), precursors formed earlier in the yolk sac (YS) display limited differentiation and self-renewal potentials. The mechanisms leading to the generation of the precursors in both sites are still largely unknown, as are the molecular basis underlying their different potential. A possible approach to assess the role of candidate genes is to transfer or modulate their expression/activity in both sites. We thus designed and compared transduction protocols to target either native extra-embryonic precursors, or hematopoietic precursors.</p> <p>Results</p> <p>One transduction protocol involves transient modification of gene expression through <it>in situ </it>electroporation of the prospective blood islands, which allows the evolution of transfected mesodermal cells in their "normal" environment, upon organ culture. Following <it>in situ </it>electroporation of a GFP reporter construct into the YS cavity of embryos at post-streak (mesodermal/pre-hematopoietic precursors) or early somite (hematopoietic precursors) stages, high GFP expression levels as well as a good preservation of cell viability is observed in YS explants. Moreover, the erythro-myeloid progeny typical of the YS arises from GFP⁺mesodermal cells or hematopoietic precursors, even if the number of targeted precursors is low. The second approach, based on retroviral transduction allows a very efficient transduction of large precursor numbers, but may only be used to target 8 dpc YS hematopoietic precursors. Again, transduced cells generate a progeny quantitatively and qualitatively similar to that of control YS.</p> <p>Conclusion</p> <p>We thus provide two protocols whose combination may allow a thorough study of both early and late events of hematopoietic development in the murine YS. <it>In situ </it>electroporation constitutes the only possible gene transfer method to transduce mesodermal/pre-hematopoietic precursors and analyze the earliest steps of hematopoietic development. Both <it>in situ </it>electroporation and retroviral transduction may be used to target early hematopoietic precursors, but the latter appears more convenient if a large pool of stably transduced cells is required. We discuss the assets and limitation of both methods, which may be alternatively chosen depending on scientific constraints.</p