This is similar to the levels of reconstitution we achieved with the cultured progeny of 12 25,000 cellscells+ cells.
It should also be pointed out that secondary transplantation showed roughly equivalent levels of reconstitution from primary recipients with single or compound mutants.
Where CD34+ cells derived from adults were used much lower levels of reconstitution were achieved, possibly reflecting reduced HSC potential with increasing donor age.
When transferred human cells were CD34-selected, significant levels of reconstitution (0.12 0.88%) were observed in all secondary recipients in three separate experiments (Table 1).
Similar levels of reconstitution were detected in NOD/Scid mice that received grafts as adults but no CD45+ cells were present in adult BALB/c Rag2−/−/γc−/− recipient mice (data not shown).
However, we observed similar levels of reconstitution (57.1% for pregnant, n = 7 and 55.6% for virgin, n = 9) suggesting that the rise in luciferase expressing CD24+CD29hicells was indeed a rise in MaSCs.
Pearson's correlation and Mann Whitney test were applied for analysis of humanized mice with different levels of reconstitution (GraphPad Prism 5).
We also found that while the PHD mutant can be phosphorylated and activate downstream signals when overexpressed at high levels, low level reconstitution of knockout cells with the PHD mutant results in lack of MEKK1 phosphorylation after vinblastine treatment.
HIS mice with a good level of human reconstitution (>20% hCD45+ cells) were immunized by intramuscular route (biceps femoris) using a 29G needle, three times on weeks 14, 16 and 18 with either 100 µl of the HBV vaccine (Engerix-B, GlaxoSmithKline) or 50 µl of tetanus toxoid (TT) containing vaccine (Tetanus vaccine, The Netherlands Vaccine Institute).
Compared to B cell reconstitution, the level of T cell reconstitution in the adult NSG mice injected with expanded CD34+ cells+ cells was fairly low (Figure 4M O, Table 2).
