Fetal HSCs (Lin−/Sca-1+/c-Kit+/Flk2−/CD150+/CD48−) were isolated from the fetal livers of E14.5 Mcm3+/+, Mcm3+/Lox or Mcm3Lox/Lox CD45.2 C57Bl/6 donor mice, and transplanted into lethally irradiated CD45.1 C57Bl/6 primary recipients (50 fetal HSCs per mouse; 5 mice per group) together with 300,000 Sca-1-depleted CD45.1 helper BM cells. 4 months post-transplantation, the primary recipients were sacrificed to assess the percentage of donor-derived chimerism in the bone marrow (BM), spleen, peripheral blood and HSC compartment and to re-isolate donor-derived adult MCM3+/+ or MCM3Lox/Lox HSCs53 (link). Donor chimerism was analyzed using CD45.2-FITC, (eBioscience 12-0453-83; 1:50), B220-APC-e780 (47-0452-82; 1:800), Gr-1-PB (1:400), Mac-1-PE-Cy7 (1:3200), CD3-e660 (eBioscience, 50-0032-82; 1:400) and Ter-119-PE-Cy5 (eBioscience 15-5921-83; 1:800) antibodies. Re-isolated CD45.2+ donor-derived adult MCM3+/+ or MCM3Lox/Lox HSCs were transplanted into lethally irradiated CD45.1 secondary recipients (500 HSCs per mouse; 5 mice per group) together with 300,000 Sca-1-depleted CD45.1 helper BM cells.
Protocol detail
Characterizing Fetal HSC Function via Transplantation
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Adult
Antibodies
Blood
Bone marrow
Bone marrow cells
C57bl mice
Cd150
Chimerism
Donor
Fetal
Fitc
Hscs
Livers
Mac 1
Mice
Sca 1
Spleen
Transplantation
Corresponding Organization :
Other organizations : Spanish National Cancer Research Centre, Broad Center, Spanish National Centre for Cardiovascular Research
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Variable analysis
independent variables
- Mcm3 genotype of fetal HSCs (Mcm3+/+, Mcm3+/Lox, or Mcm3Lox/Lox)
- Percentage of donor-derived chimerism in the bone marrow (BM), spleen, peripheral blood, and HSC compartment
- Ability of re-isolated donor-derived adult Mcm3+/+ or Mcm3Lox/Lox HSCs to reconstitute lethally irradiated secondary recipients
- Age of donor mice (E14.5)
- Strain of donor and recipient mice (C57Bl/6)
- Number of fetal HSCs transplanted per primary recipient (50)
- Number of Sca-1-depleted CD45.1 helper BM cells transplanted per primary and secondary recipient (300,000)
- Irradiation of primary and secondary recipients (lethally irradiated)
- Not specified
- Not specified
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