Members of the myocyte enhancer factor-2 (MEF2) family of MADS (MCM1, agamous, deficiens, serum response factor)-box transcription factors bind an A-T-rich DNA sequence associated with muscle-specific genes. The murine MEF2C gene is expressed in heart precursor cells before formation of the linear heart tube. In mice homozygous for a null mutation of MEF2C, the heart tube did not undergo looping morphogenesis, the future right ventricle did not form, and a subset of cardiac muscle genes was not expressed. The absence of the right ventricular region of the mutant heart correlated with down-regulation of the dHAND gene, which encodes a basic helix-loop-helix transcription factor required for cardiac morphogenesis. Thus, MEF2C is an essential regulator of cardiac myogenesis and right ventricular development.The mechanisms that regulate heart formation during embryogenesis are only beginning to be elucidated (1). Members of the MEF2 family of transcription factors bind a conserved A-T-rich DNA sequence associated with most cardiac muscle structural genes (2) and are expressed in cardiogenic precursor cells and differentiated cardiomyocytes during embryogenesis (3). MEF2 factors are also expressed in skeletal and smooth muscle cell lineages (3, 4), and MEF2 binding sites are essential for expression of muscle genes in all three muscle cell types (5).There are four MEF2 genes in vertebrate species, designated MEF2A, -B, -C, and -D, share homology in an NH 2 -terminal MADS-box and an adjacent motif known as the MEF2 * To whom correspondence should be addressed. To investigate MEF2C function during mouse embryogenesis, we inactivated this gene with a targeting vector (10) that deleted the second protein-coding exon, which encodes amino acids 18 to 86 (Fig. 1). The MADS and MEF2 domains are contained in residues 1 to 56 and 57 to 86, respectively, and the residues deleted by the mutation are essential for DNA binding and dimerization (7). The vector was introduced into embryonic stem (ES) cells by electroporation, clones were isolated after positive-negative selection (11), and genomic DNA was analyzed by Southern blot analysis for gene replacement at the MEF2C locus (12). The frequency of ES cell clones bearing a targeted MEF2C allele was 1: 7. Three targeted ES cell clones were injected into blastocysts isolated from C57BL/6J mice to generate chimeras, two of which transmitted the mutation through the germ line. Mice heterozygous for the targeted allele showed no discernible phenotype and were intercrossed to obtain MEF2C homozygous null offspring. The genotypes of offspring from heterozygous intercrosses were determined within 1 to 3 weeks after birth by Southern blot analysis of DNA obtained from tail biopsies. No neonates homozygous for the MEF2C mutation were found among 189 offspring and no neonatal lethality was observed, indicating that the homozygous mutation resulted in embryonic lethality. HHS Public AccessWe determined the genotypes of embryos between E6.5 and E12.5 by Southern blot or polymerase cha...

Inducing destruction of specific mRNA using small interfering RNA (siRNA) is a powerful tool in analysis of protein function, but its use as a therapeutic modality has been limited by inefficient or impractical delivery systems. We have used siRNA incorporated into the neutral liposome 1,2-dioleoylsn-glycero-3-phosphatidylcholine (DOPC) for efficient in vivo siRNA delivery. In nude mice bearing i.p. ovarian tumors, nonsilencing siRNA tagged with the fluorochrome Alexa 555 was encapsulated into DOPC liposomes and shown to be taken up by the tumor as well as many major organs. Furthermore, DOPC-encapsulated siRNA targeting the oncoprotein EphA2 was highly effective in reducing in vivo EphA2 expression 48 hours after a single dose as measured by both Western blot and immunohistochemistry. Therapy experiments in an orthotopic mouse model of ovarian cancer were initiated 1 week after injection of either HeyA8 or SKOV3ip1 cell lines. Three weeks of treatment with EphA2-targeting siRNA-DOPC (150 Mg/kg twice weekly) reduced tumor growth when compared with a nonsilencing siRNA (SKOV3ip1: 0.35 versus 0.70 g; P = 0.020; HeyA8: 0.98 versus 1.51 g; P = 0.16). When EphA2-targeting siRNA-DOPC was combined with paclitaxel, tumor growth was dramatically reduced compared with treatment with paclitaxel and a nonsilencing siRNA (SKOV3ip1: 0.04 versus 0.22 g; P < 0.001; HeyA8: 0.21 versus 0.84 g; P = 0.0027). These studies show the feasibility of siRNA as a clinically applicable therapeutic modality. (Cancer Res 2005; 65(15): 6910-8)

The molecular diversity of receptors in human blood vessels remains largely unexplored. We developed a selection method in which peptides that home to specific vascular beds are identified after administration of a peptide library. Here we report the first in vivo screening of a peptide library in a patient. We surveyed 47,160 motifs that localized to different organs. This large-scale screening indicates that the tissue distribution of circulating peptides is nonrandom. High-throughput analysis of the motifs revealed similarities to ligands for differentially expressed cell-surface proteins, and a candidate ligand-receptor pair was validated. These data represent a step toward the construction of a molecular map of human vasculature and may have broad implications for the development of targeted therapies.

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