![Figure 7. 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+/− Dnmt3awt/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.](https://ash.silverchair-cdn.com/ash/content_public/journal/blood/125/14/10.1182_blood-2014-06-585273/4/2206f7.jpeg?Expires=1735501859&Signature=NspKFiL52jfd2dQQ5ScqhnwtrH1dM515vOis77sO0NgcBErV2AyLzbjimVNeXjpXOa3o7pR8rGQTWCxTNuntjtt5hJCkecRI8sURTi-UPS-uVwW~WR~0NMVgdnN2MxzzZDrj06JnjZUZKysin6FPqeX~uIkMl~E2OG0eeTTNd7KFzjFampCinuSbhhlSLK8ULWUZkN-zA75OJikVE-M0lvxAk093Y5pM2c65R3ext3Jjq0s~ErbRE0GREsgVEFyium8ShIC2-37OAXCrXBeIw6u9n62be8ddFFUP8IZDbSP1YBk7eFTLrkb-Lf82FBqQvIeVxWvDonBt88MjxdwWvg__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
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+/−Dnmt3awt/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.
