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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.

Giroux SJ, Alves-Leiva C, Lécluse Y, Martin P, Albagli O, Godin I - BMC Dev. Biol. (2007)

Bottom Line: Hematopoietic development in vertebrate embryos results from the sequential contribution of two pools of precursors independently generated.We thus designed and compared transduction protocols to target either native extra-embryonic precursors, or hematopoietic precursors.We discuss the assets and limitation of both methods, which may be alternatively chosen depending on scientific constraints.

View Article: PubMed Central - HTML - PubMed

Affiliation: INSERM U790, Institut Gustave Roussy-PR1, Villejuif, France. Sebastien.Giroux@u-bourgogne.fr <Sebastien.Giroux@u-bourgogne.fr>

ABSTRACT

Background: 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.

Results: One transduction protocol involves transient modification of gene expression through in situ electroporation of the prospective blood islands, which allows the evolution of transfected mesodermal cells in their "normal" environment, upon organ culture. Following in situ 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.

Conclusion: 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. In situ electroporation constitutes the only possible gene transfer method to transduce mesodermal/pre-hematopoietic precursors and analyze the earliest steps of hematopoietic development. Both in situ 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.

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In situ electroporation parameters. Left: Scheme of a square wave pulse delivered during in situ electroporation. Right: Optimal parameters used for YS in situ electroporation at the OB-EB stages.
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Figure 3: In situ electroporation parameters. Left: Scheme of a square wave pulse delivered during in situ electroporation. Right: Optimal parameters used for YS in situ electroporation at the OB-EB stages.

Mentions: We used a square-wave pulse generator (ECM830, BTX) to deliver the construct at a low voltage, thus preserving tissue integrity. The best results (efficient electroporation, preservation of explants morphology and recovery of a normal hematopoietic progeny from transfected precursors, see below) are obtained with five 50 ms pulses of 30 V at 500 ms intervals, with a 4 mm distance between the electrodes (Fig. 3). In theory, with such settings, a maximum 40–50% of the blood islands cells might take up the plasmid. In order to increase the targeted area, pulses were applied twice, with an inverted field, using the same parameter set-up. Unfortunately, the gain in the number of transduced cells was annihilated by a decrease in cell viability (Data not shown).


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.

Giroux SJ, Alves-Leiva C, Lécluse Y, Martin P, Albagli O, Godin I - BMC Dev. Biol. (2007)

In situ electroporation parameters. Left: Scheme of a square wave pulse delivered during in situ electroporation. Right: Optimal parameters used for YS in situ electroporation at the OB-EB stages.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC1941737&req=5

Figure 3: In situ electroporation parameters. Left: Scheme of a square wave pulse delivered during in situ electroporation. Right: Optimal parameters used for YS in situ electroporation at the OB-EB stages.
Mentions: We used a square-wave pulse generator (ECM830, BTX) to deliver the construct at a low voltage, thus preserving tissue integrity. The best results (efficient electroporation, preservation of explants morphology and recovery of a normal hematopoietic progeny from transfected precursors, see below) are obtained with five 50 ms pulses of 30 V at 500 ms intervals, with a 4 mm distance between the electrodes (Fig. 3). In theory, with such settings, a maximum 40–50% of the blood islands cells might take up the plasmid. In order to increase the targeted area, pulses were applied twice, with an inverted field, using the same parameter set-up. Unfortunately, the gain in the number of transduced cells was annihilated by a decrease in cell viability (Data not shown).

Bottom Line: Hematopoietic development in vertebrate embryos results from the sequential contribution of two pools of precursors independently generated.We thus designed and compared transduction protocols to target either native extra-embryonic precursors, or hematopoietic precursors.We discuss the assets and limitation of both methods, which may be alternatively chosen depending on scientific constraints.

View Article: PubMed Central - HTML - PubMed

Affiliation: INSERM U790, Institut Gustave Roussy-PR1, Villejuif, France. Sebastien.Giroux@u-bourgogne.fr <Sebastien.Giroux@u-bourgogne.fr>

ABSTRACT

Background: 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.

Results: One transduction protocol involves transient modification of gene expression through in situ electroporation of the prospective blood islands, which allows the evolution of transfected mesodermal cells in their "normal" environment, upon organ culture. Following in situ 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.

Conclusion: 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. In situ electroporation constitutes the only possible gene transfer method to transduce mesodermal/pre-hematopoietic precursors and analyze the earliest steps of hematopoietic development. Both in situ 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.

Show MeSH