High infant mortality and growth retardation in AML1-3 mice. (A) Kaplan-Meier survival curves for the wild-type–AML1–knock-in (AML1-KI), AML1-2, and AML1-3 mutants. The solid line indicates the survival curve for homozygous mutants; the broken line, heterozygous; and the dotted line, wild-type littermates. Homo indicates homozygous; and hetero, heterozygous. (B) Comparative body weight for littermates according to genotype is indicated by columns. For randomly extracted litters, mean body weight of heterozygous or homozygous mice relative to that of wild-type littermates is shown. Six AML1-KI, 8 AML1-2, and 11 AML1-3 litters were analyzed. (C) Gross appearance of representative 1-week-old AML1-2 and AML1-3 litters. Arrows indicate pups homozygous for the AML1-3 mutant. (D) Effects of the AML1-3 molecule on the AML3's trans-activation activity were examined by reporter promoter assay experiments using an AML3-dependent gene, the mouse osteocalcin gene 2 (mOG2) promoter reporter construct. A DNA fragment (-147 to +13) of the mOG2 promoter was isolated by means of PCR as described in the literature⁴⁶  and then subcloned into a firefly luciferase construct, pGL3-basic (pGL3-mOG2). Relative activities of the various combinations of AML3-related effector plasmids on the pGL3-mOG2 in the presence of exogenous CBFβ in HeLa cells are indicated by the columns (see “Materials and methods”). Bars signify standard deviation. This mOG2 promoter DNA fragment contained one binding site for AML3,^46,47  and AML3 molecule exerted a somewhat weak but reproducibly consistent activity on this promoter construct (lanes 2-4), as previously reported for nonosseous cells.^46,47  Addition of AML1-3 had no obvious effects on this AML3's trans-activation activity (lanes 11-13), whereas addition of AML1b or AML1-2 to AML3 appeared to augment the promoter activity (lanes 5-10). Three independent experiments were performed and similar results were obtained. (E) Semiquantitative RT-PCR for AML3-dependent genes^{39,40,46,48,49}  was performed for AML1-3 embryos. Total RNA samples were carefully isolated from maxillae, mandibles, and calvariae of E18 embryos from intercrossed AML1-3 heterozygotes as described.⁴⁸  Expression levels comparable to those of the controls were observed in the RNA samples from AML1-3 homozygous animals (lanes 11-15) for the genes examined, including bone sialoprotein (BSP), collagen I alpha 1 (Col-1), osteocalcin (OCN), and osteopontin (OPN), in accordance with the standard levels for HPRT. (F) Skeleton of newborn mice homozygous for AML1-2 (left) and AML1-3 (right) mutants with their respective wild-type littermates. Skeletons were stained with Alcian blue and alizarin red. Photographs for (C) and (F) were taken with a digital camera (Coolpix 4300, Nikon) equipped with (C) or without (F) a macro-light (SL-1, Nikon).