We have introduced the nlacZ reporter gene into the locus of the myogenic factor gene myf-5 by homologous recombination in embryonic stem (ES) cells. Targeted ES clones were injected into precompaction morula, and the beta-galactosidase expression pattern was monitored. These mice permit the sensitive visualization of myf-5 expression throughout the embryo, and provide a standard for comparing it with that seen with different myf-5/nlacZ transgenes. Thus, in a comparison using ES cells in chimaeric embryos containing the targeted or randomly integrated myf-5/nlacZ construct, we demonstrate that 5.5 kbp of myf-5 upstream flanking sequence including exon1 and most of intron1 directs some skeletal muscle expression, but this is neither qualitatively nor quantitatively equivalent to that of the endogenous gene. Myf-5 is expressed early, before terminal myogenesis takes place in the medial half of the somite, and subsequently it is a major myogenic factor as skeletal muscle forms. All skeletal muscle shows beta-galactosidase activity, even after birth, indicating that myf-5 expression is not confined to primary myotubes, which are derived from embryonic myoblasts, but is also present in muscles containing different adult fibre types. The presence of myf-5 transcripts from the endogenous gene in older muscle was confirmed by in situ hybridization. These results suggest that the myf-5 gene is not activated in only a subset of muscle cells and are consistent with the results on the MyoD knockout mice.

We have obtained transgenic mice expressing nuclearly targeted beta‐galactosidase (nls‐beta‐gal) under the control of a chicken acetylcholine receptor alpha‐subunit promoter. The expression of the transgene was detected in early somites, starting before embryonic day 9.5. In 13‐day embryos, the expression pattern of the transgene closely paralleled that of the endogenous mouse alpha‐subunit gene, assessed by in situ hybridization. Our results illustrate, with single‐cell resolution, the tissue specificity of this alpha‐subunit promoter during embryogenesis. After birth, the overall beta‐galactosidase activity rapidly decreased with age. However, in diaphragms of newborn animals, beta‐galactosidase activity selectively persisted in nuclei underlying the motor endplates. The latter were revealed by an acetylcholinesterase stain. Nls‐beta‐gal was also visualized by indirect immunofluorescence, while endplates were labelled with fluorescent alpha‐bungarotoxin. Confocal microscopy unambiguously identified the more intensely stained nuclei as synaptic ‘fundamental nuclei’, and allowed estimates of relative staining levels. Thus an 842 bp acetylcholine receptor gene promoter confers preferential synaptic expression to a reporter gene within myofibres in vivo.

Matings of female DDK mice with males of the BALB/c strain are sterile, whereas reciprocal crosses are normally fertile. We used nuclear transplantation between the hybrid eggs of these two strains to investigate the basis of this effect. We demonstrate that the observed sterility results from early embryonic mortality, that the mortality is due to a modification of the egg cytoplasm, and that the modification is mediated by the male pronucleus. Once established, this modification may affect female pronuclei of unrelated genotype such as C57BL/6. These results support the notion that a product derived from the male genome acts at the pronuclear stage and can affect later stages of embryonic development.When female DDK mice are mated to males of other strains, they generally exhibit low fertility, whereas intrastrain matings or reciprocal crosses (of DDK males with females of other strains) show normal fertility (1). This phenomenon, which is due to death occurring before or soon after implantation, stems from a particularity of the embryos themselves and not from an adverse effect of the DDK uterine environment (2). Genetic analysis (3) has suggested that four alleles at either one locus or two closely linked loci are involved in the establishment of an incompatibility between the cytoplasm of egg and a factor in the sperm. Here we examine directly whether such an interaction determines the appearance of this embryonic lethality. We performed reciprocal nuclear transplantations (4) between one-cell embryos produced from matings involving DDK, BALB/c, and C57BL/6 mice. Our results show that a BALB/c male pronucleus can modify the DDK egg cytoplasm in a way that leads to early embryonic lethality. MATERIALS AND METHODSEmbryo Isolation. Embryos were obtained from DDK, BALB/c, and C57BL/6 strains of mice bred at the Pasteur Institute and maintained on a 12-hr light-dark cycle. Prepuberal females (4-6 weeks old) were induced to superovulate by intraperitoneal injection of pregnant mare serum gonadotropin (Folligon-Intervet, 2.5 international units) followed by human chorionic gonadotropin (Chorulon-Intervet, 5 international units) 44-49 hr later and then caged overnight with males. One-cell-stage embryos were isolated 19-26 hr after human chorionic gonadotropin injection from females with vaginal plugs (day 1). The ampullary region of excised oviducts was placed at 30°C in PB1 medium (5) containing bovine serum albumin (Sigma catalog no. A 7638) at 4 mg/ml together with bovine hyaluronidase (Sigma) at 500 units/ml and punctured with watchmaker forceps to release the clutch of eggs. After 3-5 min the cumulus cells dissociated and the eggs were washed by transfer through successive passages (four to six) in fresh PB1 medium. After careful examination under a stereo microscope (x 80) fertilized eggs showing two pronuclei and polar body were pooled from several females of the same genotype.In Vitro Culture. Embryos were cultured under paraffin oil (BDH) in 10-A.l drops of Whitten's medium (6) in an atmosphere of ...

In vivo, the steady-state level of c-myc mRNA is mainly controlled by posttranscriptional mechanisms. Using a panel of transgenic mice in which various versions of the human c-myc proto-oncogene were under the control of major histocompatibility complex H-2K b class I regulatory sequences, we have shown that the 5 and the 3 noncoding sequences are dispensable for obtaining a regulated expression of the transgene in adult quiescent tissues, at the start of liver regeneration, and after inhibition of protein synthesis. These results indicated that the coding sequences were sufficient to ensure a regulated c-myc expression. In the present study, we have pursued this analysis with transgenes containing one or the other of the two c-myc coding exons either alone or in association with the c-myc 3 untranslated region. We demonstrate that each of the exons contains determinants which control c-myc mRNA expression. Moreover, we show that in the liver, c-myc exon 2 sequences are able to down-regulate an otherwise stable H-2K mRNA when embedded within it and to induce its transient accumulation after cycloheximide treatment and soon after liver ablation. Finally, the use of transgenes with different coding capacities has allowed us to postulate that the primary mRNA sequence itself and not c-Myc peptides is an important component of c-myc posttranscriptional regulation.In recent years, it has been realized that the rates of RNA decay in the nucleus and in the cytoplasm are important in determining the level of expression of a gene (reviewed in reference 5). In eukaryotic cells, the range of mRNA stability can vary over several orders of magnitude, and numerous studies have indicated that several mRNAs contain within them the information necessary to determine their stability. Transiently expressed genes such as those encoding proto-oncogenes and growth factors have far shorter half-lives than others, such as those encoding ␤-globin or albumin (15,32). The mechanisms of differential degradation are an attractive problem which covers several distinct aspects, such as analysis of the structural features of mRNA that determine its susceptibility to decay, identification of the locations of the destabilizing determinants, determination of the trans-acting factors with which they interact, and discovery of the physiological signals which alter rates of mRNA decay (reviewed in reference 30).