Dnmt3a dysregulation mediates miR-29a/b-1-deficient HSC defects. (A) shRNAs (197 and 6567) efficiently knock down Dnmt3a mRNA levels by qRT-PCR in NIH 3T3 cells. (B) Knock down of Dnmt3a partially restores miR-29a/b-1-null HSPC function as assessed by methylcellulose colony formation assay. Bone marrow cells were harvested from fluorouracil (5-FU) treated mice and infected with either empty GFP⁺ retrovirus or shRNAs against Dnmt3a. GFP⁺ cells were double-sorted after 2 rounds of infection and plated in 6-well plates with 200 cells per well, and colony numbers were counted at day 12. (C) The 3- to 4-week-old progeny from miR-29a^+/−Dnmt3a^wt/fl[Mx1-Cre] mice were treated with poly I:C 3 times every other day and were analyzed 1 month following the last injection. LSK (left) cells were significantly increased in miR-29a^+/−;Dnmt3^+/− mice, but committed progenitors were not affected (right). (D) Evaluation of cell cycle status of LSK cells in miR-29a^+/−, Dnmt3a^+/−, and miR-29a^+/−;Dnmt3a^+/− mice using a DAPI DNA stain. Shown with representative flow cytometry analysis (left) and summarized results (right). (E) Five million total bone marrow cells from miR-29a^+/+;Dnmt3a^+/+, miR-29a^+/−;Dnmt3a^+/+, miR-29a^+/+;Dnmt3a^+/−, and miR-29a^+/−;Dnmt3a^+/− mice were transplanted into lethally irradiated congenic recipients with 5 million competitor bone marrow cells. Peripheral blood donor chimerism was determined 4 months after transplant. (F) Bone marrow HSPCs were analyzed for donor cell chimerism. Error bars represent SEM from 5 to 12 mice for all experiments shown. Statistical significance was calculated using a Student t test: *P < .05; **P < .01.