Hybrid vigor, fetal overgrowth, and viability of mice derived by nuclear cloning and tetraploid embryo complementation.

To assess whether heterozygosity of the donor cell genome was a general parameter crucial for long-term survival of cloned animals, we tested the ability of embryonic stem (ES) cells with either an inbred or F(1) genetic background to generate cloned mice by nuclear transfer. Most clones derived from five F(1) ES cell lines survived to adulthood. In contrast, clones from three inbred ES cell lines invariably died shortly after birth due to respiratory failure. Comparison of mice derived from nuclear cloning, in which a complete blastocyst is derived from a single ES cell, and tetraploid blastocyst complementation, in which only the inner cell mass is formed from a few injected ES cells, allows us to determine which phenotypes depend on the technique or on the characteristics of the ES cell line. Neonatal lethality also has been reported in mice entirely derived from inbred ES cells that had been injected into tetraploid blastocysts (ES cell-tetraploids). Like inbred clones, ES cell-tetraploid pups derived from inbred ES cell lines died shortly after delivery with signs of respiratory distress. In contrast, most ES cell-tetraploid neonates, derived from six F(1) ES cell lines, developed into fertile adults. Cloned pups obtained from both inbred and F(1) ES cell nuclei frequently displayed increased placental and birth weights whereas ES cell-tetraploid pups were of normal weight. The potency of F(1) ES cells to generate live, fertile adults was not lost after either long-term in vitro culture or serial gene targeting events. We conclude that genetic heterozygosity is a crucial parameter for postnatal survival of mice that are entirely derived from ES cells by either nuclear cloning or tetraploid embryo complementation. In addition, our results demonstrate that tetraploid embryo complementation using F(1) ES cells represents a simple, efficient procedure for deriving animals with complex genetic alterations without the need for a chimeric intermediate.

Loss of genomic methylation causes p53-dependent apoptosis and epigenetic deregulation.

Cytosine methylation of mammalian DNA is essential for the proper epigenetic regulation of gene expression and maintenance of genomic integrity. To define the mechanism through which demethylated cells die, and to establish a paradigm for identifying genes regulated by DNA methylation, we have generated mice with a conditional allele for the maintenance DNA methyltransferase gene Dnmt1. Cre-mediated deletion of Dnmt1 causes demethylation of cultured fibroblasts and a uniform p53-dependent cell death. Mutational inactivation of Trp53 partially rescues the demethylated fibroblasts for up to five population doublings in culture. Oligonucleotide microarray analysis showed that up to 10% of genes are aberrantly expressed in demethylated fibroblasts. Our results demonstrate that loss of Dnmt1 causes cell-type-specific changes in gene expression that impinge on several pathways, including expression of imprinted genes, cell-cycle control, growth factor/receptor signal transduction and mobilization of retroelements.

Marking IL-4-producing cells by knock-in of the IL-4 gene.

IL-4 is a cytokine which can be expressed by a number of cell types including Th2 cells, mast cells and a population of CD4+ NK1.1+ NK T cells. Although phenotypic markers exist for identifying each of these cell types, there is at present no known cell surface marker common to all IL-4-producing cells. Using gene targeting in embryonic stem cells, we have modified the IL-4 locus by knock-in of a transmembrane domain to generate mice that express a membrane-bound form of IL-4 (mIL-4). Flow cytometry using an IL-4-specific mAb allowed the detection of IL-secreting Th2 cells, mast cells and NK T cells from mIL-4 mice. Furthermore, the analysis of immune responses in mIL-4 mice following immunization with anti-CD3 and anti-IgD has allowed us to identify distinct subpopulations of IL-4-producing NK T cells. Thus, the expression of IL-4 in a membrane-bound form provides a novel method for the identification and characterization of IL-4-producing cells.

Decreased neonatal dietary fat absorption and T cell cytotoxicity in pancreatic lipase-related protein 2-deficient mice.

The pancreas secretes several different lipases. The most abundant is pancreatic triglyceride lipase (PTL). The pancreas also synthesizes two homologues of PTL, the pancreatic lipase-related proteins 1 and 2 (PLRP1 and PLRP2). Cytotoxic T-lymphocytes also express PLRP2 under certain conditions. We sought to determine the role of PLRP2 in fat absorption and in T-cell cytotoxicity by creating a PLRP2-deficient mouse. Adult PLRP2-deficient mice had normal fat absorption. In contrast, suckling PLRP2-deficient mice had fat malabsorption evidenced by increased fecal weight, increased fecal fats, and the presence of undigested and partially digested dietary triglycerides in the feces. As a result, the PLRP2-deficient pups had a decreased rate of weight gain. To assess T cell cytotoxicity, we immunized PLRP2-deficient mice with a mastocytoma cell line, P815, and determined the ability of splenocytes from the immunized mice to kill P815 cells in a 51Cr release assay. PLRP2-deficient cells had deficient killing activity in this assay, and PLRP2-deficient splenocytes released fewer fatty acid from the target cells than did control cells. Our results provide the first evidence of a physiological function for PLRP2. PLRP2 participates in T cell cytotoxicity, and PLRP2 performs a crucial role in the digestion of dietary fats in suckling animals.

DNA hypomethylation leads to elevated mutation rates.

Genome-wide demethylation has been suggested to be a step in carcinogenesis. Evidence for this notion comes from the frequently observed global DNA hypomethylation in tumour cells, and from a recent study suggesting that defects in DNA methylation might contribute to the genomic instability of some colorectal tumour cell lines. DNA hypomethylation has also been associated with abnormal chromosomal structures, as observed in cells from patients with ICF (Immunodeficiency, Centromeric instability and Facial abnormalities) syndrome and in cells treated with the demethylating agent 5-azadeoxycytidine. Here we report that murine embryonic stem cells nullizygous for the major DNA methyltransferase (Dnmt1) gene exhibited significantly elevated mutation rates at both the endogenous hypoxanthine phosphoribosyltransferase (Hprt) gene and an integrated viral thymidine kinase (tk) transgene. Gene deletions were the predominant mutations at both loci. The major cause of the observed tk deletions was either mitotic recombination or chromosomal loss accompanied by duplication of the remaining chromosome. Our results imply an important role for mammalian DNA methylation in maintaining genome stability.

The role of a single formin isoform in the limb and renal phenotypes of limb deformity.

BACKGROUND: Mutations of the murine limb deformity (ld) locus are responsible for a pleiotropic phenotype of completely penetrant limb malformations and incompletely penetrant renal agenesis and/or dysgenesis. The ld locus encodes a complex family of mRNA and protein isoforms. MATERIALS AND METHODS: To examine the role of one of the more prominent of these isoforms, isoform IV, we specifically eliminated it by gene targeting. RESULTS: Unlike other mutant ld mice, homozygous mice bearing this isoform IV disruption display incompletely penetrant renal agenesis, but have perfectly normal limbs. Whole mount in situ hybridization demonstrated that this targeted disruption was specific for isoform IV and did not interfere with the expression of other ld isoforms. The isoform IV-disrupted allele of ld does not complement the renal agenesis phenotype of other ld alleles, in a manner consistent with its penetrance, and like the isoform IV-deficient mice, these compound heterozygotes have normal limbs. Sequence analysis of formin isoform IV in other ld mutant alleles did not detect any amino acid changes relative to the strain of origin of the mutant allele. CONCLUSIONS: Thus, the disruption of isoform IV is sufficient for the renal agenesis phenotype, but not the limb phenotype of ld mutant mice. Structural mutations in this isoform are only one of several genetic mechanisms leading to the renal phenotype, since amino acid changes in this isoform were not detected. These results demonstrate that this gene is limb deformity, and that variable isoform expression may play a role in generating the pleiotropic ld phenotype.

Mutagenicity of 5-aza-2'-deoxycytidine is mediated by the mammalian DNA methyltransferase.

The cytosine analog 5-aza-2'-deoxycytidine has been used clinically to reactivate genes silenced by DNA methylation. In particular, patients with beta-thalassemia show fetal globin expression after administration of this hypomethylating drug. In addition, silencing of tumor suppressor gene expression by aberrant DNA methylation in tumor cells may potentially be reversed by a similar regimen. Consistent with its function in maintaining tumor suppressor gene expression, 5-aza-2'-deoxycytidine significantly reduces intestinal tumor multiplicity in the predisposed Min mouse strain. Despite its utility as an anti-cancer agent, the drug is highly mutagenic by an unknown mechanism. To gain insight into how 5-aza-2'-deoxycytidine induces mutations in vivo, we examined the mutational spectrum in an Escherichia coli lac I transgene in colonic DNA from 5-aza-2'-deoxycytidine-treated mice. Mutations induced by 5-aza-2'-deoxycytidine were predominantly at CpG dinucleotides, which implicates DNA methyltransferase in the mutagenic mechanism. C:G-->G:C transversions were the predominant class of mutations observed. We suggest a model for how the mammalian DNA methyltransferase may be involved in facilitating these mutations. The observation that 5-aza-2'-deoxycytidine-induced mutations are mediated by the enzyme suggests that novel inhibitors of DNA methyltransferase, which can inactivate the enzyme before its interaction with DNA, are needed for chemoprevention or long term therapy.

Experimental manipulation of genomic methylation.

Cytosine methylation is an important mechanism of gene regulation in mammals. Mouse embryos with reduced DNA methylation due to targeted disruption of the DNA methyltransferase gene show deregulated expression of imprinted genes. Loss of imprinting associated with loss of allele-specific methylation is one example of an epigenetic alteration found in tumor cells. Changes in DNA methylation may also be associated with facilitating protooncogene expression and inactivating tumor suppressor genes. However, cytosine methylation has additional deleterious consequences for the genome as well. CpG dinucleotides, the target of DNA methylation, are five-fold underpresented in the genome due to the high mutability of methylated cytosine. C-T transition mutations resulting from deamination of 5-methylcytosine are involved in both genetic disease and cancer. Lastly, aberrant DNA methylation may promote the genetic instability of a chromosomal locus. We review the genetic and epigenetic roles for DNA methylation during tumorigenesis gleaned from altered methycytosine patterns in tumor cells, and from pharmacologic, dietary or genetic manipulation of DNA methylation levels.

Germ-line passage is required for establishment of methylation and expression patterns of imprinted but not of nonimprinted genes.

Embryonic stem (ES) cells homozygous for a disruption of the DNA (cytosine-5)-methyltransferase gene (Dnmt) proliferate normally with their DNA highly demethylated but die upon differentiation. Expression of the wild-type Dnmt cDNA in mutant male ES cells caused an increase in methylation of bulk DNA and of the Xist and Igf2 genes to normal levels, but did not restore the methylation of the imprinted genes H19 and Igf2r. These cells differentiated normally in vitro and contributed substantially to adult chimeras. While the Xist gene was not expressed in the remethylated male ES cells, no restoration of the normal expression profile was seen for H19, Igf2r, or Igf2. This indicates that ES cells can faithfully reestablish normal methylation and expression patterns of nonimprinted genes but lack the ability to restore those of imprinted genes. Full restoration of monoallelic methylation and expression was imposed on H19, Igf2, and Igf2r upon germ-line transmission. These results are consistent with the presence of distinct de novo DNA methyltransferase activities during oogenesis and spermatogenesis, which specifically recognize imprinted genes but are absent in the postimplantation embryo and in ES cells.

Suppression of intestinal neoplasia by DNA hypomethylation.

We have used a combination of genetics and pharmacology to assess the effects of reduced DNA methyltransferase activity on ApcMin-induced intestinal neoplasia in mice. A reduction in the DNA methyltransferase activity in Min mice due to heterozygosity of the DNA methyltransferase gene, in conjunction with a weekly dose of the DNA methyltransferase inhibitor 5-aza-deoxycytidine, reduced the average number of intestinal adenomas from 113 in the control mice to only 2 polyps in the treated heterozygotes. Hence, DNA methyltransferase activity contributes substantially to tumor development in this mouse model of intestinal neoplasia. Our results argue against an oncogenic effect of DNA hypomethylation. Moreover, they are consistent with a role for DNA methyltransferase in the generation of the C to T transitions seen at high frequency in human colorectal tumors.

Formins: phosphoprotein isoforms encoded by the mouse limb deformity locus.

Mutations at the mouse limb deformity (ld) locus result in defects of growth and patterning of the limb and kidney during embryonic development. The gene responsible for this phenotype is large and complex, with the capacity to generate a number of alternatively spliced messenger RNA transcripts encoding nuclear protein isoforms called "formins." We have made polyclonal antibodies to specific formin peptides and have confirmed the authenticity of the antibodies' reactivity, using cell lines derived from mice with molecularly defined mutations at the ld locus. In addition, we have used these antibodies to detect and characterize polypeptides encoded by both wild-type and mutant ld alleles. In so doing, we show that a formin isoform (i) is modified by posttranslational phosphorylation at serine and threonine residues and (ii) when present in a crude nuclear extract, is retained by DNA-cellulose.

Placental-specific expression from the mouse placental lactogen II gene promoter.

The gene for mouse placental lactogen II (mPL-II) has been isolated and characterized. This gene contains five exons, with a transcription start site 59 nucleotides upstream of the translation initiation ATG. Introduction of a DNA construct containing 2.7 kilobases of sequence upstream of the mPL-II transcription initiation site directed the synthesis of a linked coding region for the simian virus 40 large and small tumor antigens in placental trophoblast giant cells of transgenic mice. The pattern of simian virus 40 transgene expression in the placenta was indistinguishable from that of the endogenous mPL-II gene. In contrast, the first 569 base pairs upstream of the transcription start site proved insufficient to direct placental expression. Thus, one or more elements required for placental trophoblast giant cell expression have been localized to a region between -2700 and -569 of the mPL-II gene.

The same genomic region is disrupted in two transgene-induced limb deformity alleles.

Mutations of the mouse limb deformity locus, ld, map to Chromosome (Chr) 2 and result in defects in the morphogenesis and patterning of the limb and kidney. Complementation studies have defined the existence of five recessive ld alleles. Remarkably, two of these, ldTgHd and ldTgBri, are transgene-induced mutations. Recovery of the first transgene insertional allele, ldTgHd, facilitated the molecular cloning of a large (greater than 200 kb) candidate gene at the ld locus. This gene is broadly transcribed and encodes a set of novel protein isoforms, termed formins. Here we present characterization of the ldTgBri mutation that supports the molecular identification of the ld gene. We show that the ldTgBri fails to complement both the ldTgHd and the ldOR alleles and that it has undergone a genomic deletion that disrupts the cloned ld gene and its transcripts. Curiously, the ldTgBri deletion encompasses the same 11-kb interval in which the ldTgHd insertion occurred and in which a chromosomal rearrangement has been identified in a third allele, ldIn2. These findings suggest that this region of the ld gene is a preferential site for illegitimate recombination.

A variant limb deformity transcript expressed in the embryonic mouse limb defines a novel formin.

The formins constitute a set of protein isoforms encoded by the alternatively spliced transcripts arising from the limb deformity (ld) locus of the mouse. Mutations in this locus disrupt formation of the anteroposterior axis of the embryonic limb. Although ld transcripts are widely expressed during embryogenesis, we have identified a novel transcript that is expressed in the mesenchyme and apical ectodermal ridge of the developing limb. This pattern of expression coincides with the earliest morphological defects observed in ld mutant limb buds. Moreover, the formin encoded by this transcript bears a highly acidic amino terminus, as distinguished from the basic amino terminus encoded by other ld transcripts suggesting that it may have a distinct biochemical function.

Selective expression of PDGF A and its receptor during early mouse embryogenesis.

Murine homologs of the PDGF A, PDGF B, and PDGF receptor alpha subunit genes were cloned. These were used, together with a mouse PDGF receptor beta subunit cDNA clone, to monitor gene expression in early postimplantation mouse embryos and in F9 embryonal carcinoma cells. RNAse protection analysis shows that PDGF A chain, but not B chain, mRNA is expressed in 6.5- to 8.5-day embryonic and extraembryonic tissues. Both alpha and beta receptor subunit mRNAs are expressed in early embryos, however, alpha subunit mRNA appears earlier and is more abundant than beta subunit mRNA. Undifferentiated F9 embryonal carcinoma stem cells express abundant levels of A chain, but not B chain, mRNA. Neither of the PDGF receptor genes is expressed in stem cells. Treatment with retinoic acid stimulates expression of both PDGF receptor genes. As in postimplantation mouse embryos, alpha receptor subunit mRNA appears earlier and is substantially more abundant than beta subunit mRNA. Collectively, these data demonstrate that the genes encoding the two chains of PDGF and their receptors are regulated independently during development and suggest that the two systems have some nonoverlapping functions in vivo. PDGF A, but not PDGF B, may be particularly important in modulating early events in mouse embryonic development.

The limb deformity gene is required for apical ectodermal ridge differentiation and anteroposterior limb pattern formation.

To gain insight into the role of the limb deformity (ld) gene in limb morphogenesis, we examined the morphologic details of early embryonic limb formation in the mutant ld/ld mouse. Initial morphological differences between wild-type and homozygous ld embryos are apparent during early gestational day 10, a time period during which anteroposterior limb morphogenesis occurs. As a result of a shortened anteroposterior axis, the mutant limb bud appears more pointed than its wild-type counterpart. In addition, the apical ectodermal ridge (AER), a structure crucial to both proximodistal and anteroposterior limb development, fails to differentiate properly in mutant ld embryos. Consistent with these observations, molecular analysis of the limb promordia shows that the limb ectoderm contains a level of ld transcripts fivefold higher relative to its mesenchyme. Furthermore, expression of ld transcripts in other parts of the developing embryo and in primitive streak embryos (gestational day 7) suggests possible roles for this gene in the earliest determinative events of morphogenesis. These data lead us to conclude that ld gene products are required for both proper AER differentiation and anteroposterior pattern formation in limb mesenchyme.

Gene activation and DNA binding by Drosophila Ubx and abd-A proteins.

The Ubx and abd-A gene products are required for proper development of thoracic and abdominal structures in Drosophila. We expressed LexA-Ubx and LexA-abdA fusion proteins in yeast. These proteins activated expression of target genes that carried either upstream LexA operators or upstream Ubx binding sites. Both proteins contain homeodomains. Experiments with mutant fusion proteins show that the homeodomain is not required for the proteins to form dimers or enter the nucleus, and that, when DNA binding is provided by the LexA moiety, the homeodomain is not required for gene activation. Our results suggest that the homeodomain is necessary for these proteins to bind Ubx sites, but that the homeodomain does not contact DNA exactly like bacterial helix-turn-helix proteins. Finally, our data suggest that gene activation by these proteins is a simple consequence of their binding to DNA, while negative gene regulation requires that these proteins act together with other Drosophila gene products.

Nerve growth factor regulates gene expression by several distinct mechanisms.

To help elucidate the mechanisms by which nerve growth factor (NGF) regulates gene expression, we have identified and studied four genes (a-2, d-2, d-4, and d-5) that are positively regulated by NGF in PC12 cells, including one (d-2) which has previously been identified as a putative transcription factor (NGF I-A). Three of these genes, including d-2, were induced very rapidly at the transcriptional level, but the relative time courses of transcription and mRNA accumulation of each of these three genes were distinct. The fourth gene (d-4) displayed no apparent increase in transcription that corresponded to the increase in its mRNA, suggesting that NGF may regulate its expression at a posttranscriptional level. Thus, NGF positively regulates gene expression by more than one mechanism. These genes could also be distinguished on the basis of their response to cyclic AMP. The expression of d-2 and a-2 was increased by cholera toxin and further augmented by NGF; however, cholera toxin not only failed to increase the levels of d-5 and d-4 mRNA but also actually inhibited the NGF-dependent increase. The expression of each of these genes, including d-2 (NGF I-A), was also increased by fibroblast growth factor, epidermal growth factor (EGF), phorbol myristate acetate, and in some cases insulin, showing that the regulation of these genes is not unique to NGF. Because each of these genes was expressed in response to phorbol myristate acetate and EGF, their expression may be necessary but is certainly not sufficient for neurite formation. The protein kinase inhibitor K-252a prevented the NGF-associated, but not the acidic FGF-associated, induction of d-2 and d-5 gene expression, suggesting that these two growth factors may regulate gene expression via different cellular pathways. The study of the regulation of the expression of these and other NGF-inducible genes should valuable new information concerning how NGF and other growth factors cause neural differentiation.

Chromosomal mapping of the prolactin/growth hormone gene family in the mouse.

The chromosomal assignments of genes in the PRL/GH family in the mouse have been determine in mouse-hamster hybrid cell lines. Mouse GH (mGH) appears to be encoded by a single copy gene located on chromosome 11 and is part of a highly conserved region between mouse chromosome 11 and human chromosome 17. All of the other genes in this hormone family, including those encoding mPRL, mouse placental lactogens I and II, and mouse proliferin and proliferin-related protein, map to chromosome 13.