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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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Plasmid injection protocol. Abbreviations: am: amnion; EPC: Ectoplacental cone; YSC: YS cavity OB-EB stage embryos are dissected from the decidua (A). The Reichert membrane is removed, while the EPC is kept in place (B). The plasmid solution is injected into the YSC, from the node region through the amnion (C). Plasmid filling of the YSC is visualised with fast green (D). Prior to pulse application, the electrodes are positioned parallel to the prospective blood islands ring (E). After electroporation, the YS is dissected from the embryo and placed in organ culture (D). Scale bar: 100 μm.
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Figure 2: Plasmid injection protocol. Abbreviations: am: amnion; EPC: Ectoplacental cone; YSC: YS cavity OB-EB stage embryos are dissected from the decidua (A). The Reichert membrane is removed, while the EPC is kept in place (B). The plasmid solution is injected into the YSC, from the node region through the amnion (C). Plasmid filling of the YSC is visualised with fast green (D). Prior to pulse application, the electrodes are positioned parallel to the prospective blood islands ring (E). After electroporation, the YS is dissected from the embryo and placed in organ culture (D). Scale bar: 100 μm.

Mentions: Targeting the YS-mesodermal/pre-hematopoietic precursors, while avoiding endoderm transduction, requires the injection of the construct into the YS cavity, since the mesoderm layer is facing this cavity while endoderm is exposed to the external environment. The embryos are freed from the decidua and Reichert's membrane (Fig. 2A, B) to allow injection. A capillary, back-filled with 1 μg/μl peGFP-C1 plasmid DNA solution is inserted into the YS cavity from the node region and through the amnios. Fast Green (0.01%) is added to the solution to visualise construct delivery. This injection mode, designed to limit plasmid release from the YS cavity, leads to the collapse of the amnion towards the ectoplacental cavity. Upon mouth driven-release of the plasmid, the amnion returns to its former position and an accurate injection can thus be visualized by the fast green filling of the YS cavity (Fig. 2C, D).


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)

Plasmid injection protocol. Abbreviations: am: amnion; EPC: Ectoplacental cone; YSC: YS cavity OB-EB stage embryos are dissected from the decidua (A). The Reichert membrane is removed, while the EPC is kept in place (B). The plasmid solution is injected into the YSC, from the node region through the amnion (C). Plasmid filling of the YSC is visualised with fast green (D). Prior to pulse application, the electrodes are positioned parallel to the prospective blood islands ring (E). After electroporation, the YS is dissected from the embryo and placed in organ culture (D). Scale bar: 100 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: Plasmid injection protocol. Abbreviations: am: amnion; EPC: Ectoplacental cone; YSC: YS cavity OB-EB stage embryos are dissected from the decidua (A). The Reichert membrane is removed, while the EPC is kept in place (B). The plasmid solution is injected into the YSC, from the node region through the amnion (C). Plasmid filling of the YSC is visualised with fast green (D). Prior to pulse application, the electrodes are positioned parallel to the prospective blood islands ring (E). After electroporation, the YS is dissected from the embryo and placed in organ culture (D). Scale bar: 100 μm.
Mentions: Targeting the YS-mesodermal/pre-hematopoietic precursors, while avoiding endoderm transduction, requires the injection of the construct into the YS cavity, since the mesoderm layer is facing this cavity while endoderm is exposed to the external environment. The embryos are freed from the decidua and Reichert's membrane (Fig. 2A, B) to allow injection. A capillary, back-filled with 1 μg/μl peGFP-C1 plasmid DNA solution is inserted into the YS cavity from the node region and through the amnios. Fast Green (0.01%) is added to the solution to visualise construct delivery. This injection mode, designed to limit plasmid release from the YS cavity, leads to the collapse of the amnion towards the ectoplacental cavity. Upon mouth driven-release of the plasmid, the amnion returns to its former position and an accurate injection can thus be visualized by the fast green filling of the YS cavity (Fig. 2C, D).

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