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PubMed2004

Mouse in red: red fluorescent protein expression in mouse ES cells, embryos, and adult animals.

Spectral variants of green fluorescent protein are widely used in live samples for a broad range of applications: from visualization of protein interactions, through following gene expression, to marking particular cells in complex tissues. Higher wavelength emissions (such as red) are preferred due to the lower background-autofluorescence in tissues (Miyawaka et al., Nat Cell Biol Suppl S1-7, 2003). Until now, however, red fluorescent proteins (RFP) have displayed toxicity in murine embryos, which has hampered its application in this model. Here we report strong expression of a recently developed RFP variant, DsRed.T3, in mouse ES cells, embryos, and adult mice. Our results show that the red fluorescent wavelength has a superior tissue penetrance compared with spectral variants of lower wavelength. Furthermore, we have generated an ES cell line and a corresponding transgenic mouse line in which red fluorescence is activated upon Cre excision. Finally, we introduced cell type-specifically expressed Cre transgenes into this Cre recombinase reporter cell line, and by using the tetraploid embryo complementation assay, we could directly verify the Cre recombinase specificity on ES cell-derived embryos/animals.

Animals↗
PubMed1984

The ability of normal mouse cells to reduce the malignant potential of transformed mouse bladder epithelial cells depends on their somatic origin.

Somatic cell hybrids have been made between a transformed mouse bladder carcinoma cell line and normal mouse bladder epithelium and mesenchyme. In the epithelial tumour/mesenchyme hybrids the malignant phenotype was expressed dominantly whereas in the carcinoma/normal epithelium hybrids the malignant potential was greatly reduced. In both cases the dominant in vitro and in vivo phenotype was that of the normal parental cells. All hybrid tumours were first palpable after 4-7 days, demonstrating that the tumours had not arisen as a result of in vivo selection of a sub-population of tumorigenic cells. Chromosome analysis showed that the carcinoma/normal epithelium hybrids were all in the hypertetraploid range but the large variation in the karyotypic profile of each hybrid made it impossible to implicate any specific chromosomes in the control of expression of the malignant phenotype. During normal development in bladder epithelium, terminal differentiation is associated with tetraploid formation by cell fusion. The reduction in malignancy of the carcinoma/normal epithelium hybrids may perhaps be due to the expression of genes associated with normal terminal differentiation after cell fusion and tetraploid formation. This is also supported by the more differentiated phenotype of the hybrid tumours. Of the 10 mesenchyme/epithelium hybrids analysed cytogenetically , four were in the hypertetraploid range from which little meaningful data could be obtained about specific chromosome losses. Chromosome analysis of the cells from the near-tetraploid hybrids showed only minor differences from what might have been expected from the input of the two parents; these differences appeared to be due to random chromosome loss. The maximum number of chromosomes lost from any of the hybrids was five, although one, two or three was more usual. The only consistent chromosome loss was of a single copy of chromosome 4, which in two of the hybrids represented the only chromosome change. The possibility that this loss might facilitate re-expression of the malignant phenotype is discussed.

Animals↗
PubMed1988

Mouse monoclonal antibodies detecting disialogangliosides on mouse and human T lymphomas.

Six monoclonal antibodies (MAbs) (4 IgG3 and 2 IgM) were produced by hybridomas obtained from A/J mice immunized with EL4(C57BL/6 derived-T lymphoma). They were found to react with antigens expressed on both mouse and human T-lymphomas but not on B lymphomas or normal cells. All of these antibodies reacted with the disialoganglioside GD2, GalNAc beta I----4(NeuAc alpha 2----8NeuAc alpha 2----3)Gal beta I----4Glc-Cer, by 3 different assay systems including the immune adherence inhibition test, enzyme-linked immunosorbent assay, and enzyme immunostaining on thin-layer chromatography. The binding specificities of these MAbs to disialogangliosides differed. Four MAbs (AI-201, AI-287, AI-410, and AI-425) showed restricted specificities, detecting only GD2, whereas the other 2 (AI-245 and AI-267) had a broader specificity, recognizing GD2, GD3, and GDlb. No evidence was obtained for the presence of the antigenic epitope in glycoproteins of mouse and human tumor cells. The ganglioside content of EL4 was low in comparison with that of M14 (a human melanoma cell line).

Animals↗
PubMedDate not supplied

A game of cat and mouse: xenografting of testis tissue from domestic kittens results in complete cat spermatogenesis in a mouse host.

Loss of genetic diversity because of infertility or the premature death of valuable individuals is a significant problem in the conservation of rare and endangered felid species, as well as in the maintenance of lines of cats used to study inherited feline and human disease. Attempts to overcome loss of genetic diversity have focused on freezing sperm; however, sperm cannot be collected from immature males. Previously, we reported completion of spermatogenesis in testis tissue from newborn pigs and goats grafted ectopically into host mice. The objective of this study was to extend the technique of testis tissue xenografting to the domestic cat as a model for felid species. Testes from 1- to 5-week-old domestic shorthaired kittens (n = 9) were cut into small fragments (about 0.5-1 mm3 each), and up to 8 fragments were grafted under the back skin of each castrated immunodeficient host mouse (n = 16). Histologic examination of the testis xenografts was performed between 5 and 54 weeks posttransplantation. At the time of grafting, the seminiferous cords of the donor testis tissue contained only immature Sertoli cells and gonocytes. At 14 weeks after grafting, tubular expansion was evidently caused by the proliferation of Sertoli cells and tubular lumen formation. By 18 weeks after transplantation, the seminiferous epithelium contained spermatocytes, and by 20 weeks, round spermatids were the most advanced types of germ cells. By 36 weeks after transplantation, xenografts of cat testis tissue had completed spermatogenesis. These results demonstrate the potential of xenografting to achieve full spermatogenesis in testis tissue from kittens. Therefore, sperm production in a mouse host can provide an alternative for germ line preservation from immature felids where sperm cryopreservation is not an option.

Animals↗
PubMed1991

cDNA and deduced amino acid sequence of mouse matrix gla protein: one of five glutamic acid residues potentially modified to gla is not conserved in the mouse sequence.

A cDNA library was constructed using the mouse osteoblastic cell line MC3T3-E1 treated with 1 alpha,25-dihydroxyvitamin D3, based on the finding that the treatment increased ninefold the expression of 0.7 kb matrix gla protein (MGP) mRNA. cDNA clones encoding mouse MGP were isolated from the library. The nucleotide sequence showed an open reading frame of 312 nucleotides encoding 104 amino acids. Murine MGP shared 84-89% amino acid sequence homology with bovine, rat, and human MGP. However, there are five glutamic acid residues potentially modified to gamma-carboxyglutamic acid (gla) in those species; in murine MGP, lysine replaced glutamic acid 37. Also, an extra tyrosine was added at the carboxyl terminus. The significance of the substitution is discussed in relation to the gamma-carboxylation sites in MGP protein.

Amino Acid Sequence↗
PubMed1992

Comparison of the mouse L32 ribosomal protein promoter elements in mouse myoblasts, fibers, and L cells.

The sequences required for the maximal expression of the mouse L32 ribosomal protein gene and the binding of nuclear factors to L32 promoter elements were analyzed in mouse myoblasts, fibers, and L cells. Various L32 r-protein promoter sequences were linked to the chloramphenicol acetyltransferase gene (CAT), and the expression of the chimeric genes was measured transiently or after their incorporation into the genome. The sequence requirements for maximal expression of the L32 gene are very similar among the various cells and include the previously identified L32 core promoter from approximately -150 to +75. Only the promoter regions between -45 and +11 displays significant cell type specific differences. Relative to the maximal activity in each cell type, the expression of the L32-CAT gene containing the -45 to +11 region is greater in L cells than in myoblasts or fibers. This difference is correlated with the increased activity of an L cell nuclear factor(s) that binds to this fragment. In addition, our results show that deletion of sequences between -981 and -141 causes a 50-70% reduction of the expression of the L32-CAT gene in myoblasts, fibers and L cells. The transcription of all the L32-CAT genes examined decrease after myoblasts differentiate into fibers in a manner similar to the endogenous L32 gene, but we were unable to distinguish between sequences involved in controlling the expression of the L32 gene during myoblast differentiation and those sequences required for maximal promoter activity. However, gel mobility shift assays showed differences in the binding of myoblast and fiber factors to the four promoter fragments examined. The possible role of these factor binding differences in controlling L32 transcription is discussed.

Animals↗
PubMed1983

Cell growth and differentiation in vitro in mouse macrophages transformed by a tsA mutant of simian virus 40. I. Cellular response in proliferative and phagocytic activities to the shift of temperature differs depending on the culture state in mouse bone marrow cells transformed by the tsA640 mutant of simian virus 40.

It was shown previously that mouse bone marrow cells transformed by simian virus 40 (SV40) show a reversible cell density-dependent phenotypic transition between the nonmacrophage (rapidly growing) and the macrophage (stationary) states; cells in low-density cultures are in the growing phase, express SV40 T antigen strongly as revealed by immunofluorescence, and lose typical macrophage properties such as immune phagocytosis; whereas cells in high-density cultures are in the stationary (nongrowing) phase, express SV40 T antigen weakly, and recover their macrophage properties (Takayama, 1980). In the hope of clarifying the relationship between T antigen, cell growth, and macrophage-specific cellular function, we examined the behavior at 33 and 39 degrees C of mouse bone marrow cells transformed by an SV40 gene A mutant (tsA640) whose mutation renders the molecular weight of 90K (large) T antigen temperature sensitive. The results presented in this paper suggest that functional large T antigen is required for cells in the stationary phase to initiate multiplication when transferred at lower density and is not necessary for a majority of them to maintain the nongrowing state (viability) at both high and lower cell densities, whereas it is required for cells in the growing phase to keep multiplying without losing their viability. The results also suggest that the functional large T antigen does not play a direct role in maintaining the cells as either phagocytic or nonphagocytic. It is also suggested that the physiological or tsA mutation-mediated arrest of growth may or may not be accompanied by induction and/or maintenance of cellular phagocytic activity depending on the culture state.

Animals↗
PubMed2005

Mouse HFE inhibits Tf-uptake and iron accumulation but induces non-transferrin bound iron (NTBI)-uptake in transformed mouse fibroblasts.

Iron-uptake and storage are tightly regulated to guarantee sufficient iron for essential cellular processes and to prevent the production of damaging free radicals. A non-classical class I MHC molecule, the hemochromatosis factor (HFE), has been shown to regulate iron metabolism, potentially via its interaction with the transferrin receptor. Whereas, the effect of human HFE (hHFE) on transferrin/transferrin receptor association, as well as on transferrin receptor recycling and the level of cellular iron pools in various cell lines was analyzed, very little is known about the mouse HFE (mHFE) protein. In the following study, our aim was to analyze in more detail the function of mHFE. Surprisingly, we observed that over-expression of mHFE, but not of hHFE, in a mouse transformed cell line, results in a most significant inhibition of transferrin-uptake which correlated with apoptotic cell death. mHFE inhibited transferrin-uptake immediately following transfection and this inhibition persisted in the surviving stable transfectants. Concomitantly, cellular iron derived from transferrin-iron uptake was dramatically limited. The activation of a non-transferrin bound iron-uptake pathway that functions in the stable mHFE-transfected clones could explain their normal growth curves and survival. The hypothesis that iron starvation can induce iron-uptake by a novel transferrin-independent pathway is discussed.

Animals↗
PubMed1981

Inhibition of in vitro fertilization of mouse eggs: 3-quinuclidinyl benzilate specifically blocks penetration of zonae pellucidae by mouse spermatozoa.

The fertilization in vitro of mouse with intact zonae pellucidae by mouse cauda epididymal spermatozoa was inhibited in a concentration- dependent fashion by 3-quinuclidinyl benzilate (QNB), normally used as a specific antagonist for the muscarinic class of cholinergic receptors. Inhibition was observed with both cumulus-intact and cumulus-free preparations. QNB at 50 microM inhibited fertilization of cumulus-free eggs by greater than 90% but had no effect on the fertilization of zona-free eggs. At this concentrations, QNB had no adverse effect on sperm motility, nor did it prevent binding of spermatozoa to the zona pellucida. The inhibitory effects of QNB were fully reversible. QNB is therefore a useful specific inhibitor of zona penetration. Spermatozoa in the in vitro fertilization medium bound QNB with a concentration dependence which matched that of the inhibition of fertilization. This binding was saturable and corresponded to 700 pmole/10(7) cells with KD = 10 microM. The in vitro fertilization medium contains 2% (w/v) bovine serum albumin (BSA) which also binds QNB according to the mass action law. The large amount of QNB bound to sperm in this medium appears to be QNB binding to BSA adsorbed on the sperm cell surface: these spermatozoa bind QNB specifically in the absence of BSA with a saturable capacity of only 70 fmole/10(7) cells with KD = 5 nM. Calculation of the distribution of QNB between BSA binding sites and sperm surface binding sites in the in vitro fertilization medium indicates that the specific sperm sites become saturated with the same concentration dependence as inhibition of fertilization. However, the dissociation rate of QNB from sperm in both the presence and absence of BSA is too rapid to permit confirmation of these sites as the locus of the inhibitory effect; this locus remains to be clarified.

Animals↗
PubMed1994

Onset of paternal gene activation in early mouse embryos fertilized with transgenic mouse sperm.

We investigated the onset of paternal gene expression in the early mouse embryo. We obtained transgenic mouse embryos by fertilizing BD (C57BL/6N x DBA) F1 hybrid female oocytes in vitro, with sperm from homozygous transgenic males carrying integrated chicken beta-actin promoter-driven firefly luciferase cDNA. We then examined the RNA and protein synthesis of the luciferase gene in embryos from the 1- to 2-cell stage. RNA transcripts of the luciferase gene were first detected in the 1-cell stage embryos as early as 13 hr postinsemination, just prior to elongation. By photon-count imaging, functional luciferase was identified at the 2-cell stage 23 hr postinsemination. These findings indicate that the paternal endogenous gene is already transcribed in the late 1-cell embryos, although paternally derived protein is not synthesized until the 2-cell stage. Therefore, these results suggest that the embryonic gene is activated as early as the late 1-cell stage.

Actins↗
PubMed1987

Strontium supports capacitation and the acrosome reaction in mouse sperm and rapidly activates mouse eggs.

Extracellular Ca2+ is required for capacitation and fertilization in the mouse, but very little is known about the ability of other divalent cations to substitute for Ca2+. In this study, Sr2+, Ba2+, and Mg2+ were evaluated for their ability to support capacitation, the acrosome reaction, hyperactivated motility, and fertilization. Ba2+ proved to be ineffective, but Mg2+-containing medium was able to support capacitation to a greater extent than unsupplemented Ca2+-deficient media; despite this, Ca2+ was required for fertilization. In contrast, Sr2+ proved capable of substituting for Ca2+ in all events. Furthermore, Sr2+-induced responses were indistinguishable from the corresponding Ca2+-induced ones: Sperm capacitated at the same rate and underwent the acrosome reaction to the same extent. However, demonstration of sperm:egg fusion in Sr2+ required the use of zona-free eggs. This was due not to the inability of the sperm to penetrate the zona but to the very rapid activation and cortical granule release by eggs in response to Sr2+. When zona-intact eggs were used, the block to polyspermy had been mounted by the time sperm had penetrated the zona. A 15 min exposure to Sr2+ was sufficient to block sperm fusion, but a longer exposure was required to ensure the resumption of meiosis in eggs; such a response was surprising in that the eggs were freshly ovulated and not susceptible to activation by many different treatments. Thus Sr2+ can profoundly affect both gametes in the mouse: It substitutes completely for Ca2+ in sperm responses and rapidly activates eggs, possibly by displacing Ca2+ from intracellular stores into the cytoplasm, where the Ca2+ can then trigger the various events of activation.

Acrosome↗
PubMed2004

Mouse RanBPM is a partner gene to a germline specific RNA helicase, mouse vasa homolog protein.

Germ cell-specific ATP-dependent RNA helicase, the product of the mouse vasa homolog (Mvh), has been shown to play an essential role in the development of the male germ cell. In male Mvh knockout mice, premeiotic germ cells arrest at the zygotene stage. To investigate the role of MVH protein in the progression of meiosis, we searched for genes encoding partners that interact with MVH in testicular germ cells. Using the yeast two-hybrid system, we found that MVH interacts with mouse RanBPM, a Ran-GTP binding protein involved in microtubule nucleation. RanBPM is predominantly expressed in the testis, especially in maturating spermatocytes. Within the cell, RanBPM and MVH are closely associated with perinuclear RNA-protein complexes and chromatoid bodies. The interaction of MVH with RanBPM points to a functional relationship between translational regulation and the microtubule nucleation during meiosis. Mol. Reprod. Dev. 66: 1-7, 2004.

Adaptor Proteins, Signal Transducing↗
PubMed1995

Mutagenic activation of 3-methoxy-4-aminoazobenzene by mouse renal cytochrome P450 CYP4B1: cloning and characterization of mouse CYP4B1.

A new P450 responsible for mutagenic activation of 3-methoxy-4-aminoazobenzene (3-MeO-AAB) which is a potent procarcinogen was purified from renal microsomes of male mice using an index of umu gene expression. The purified P450 had high bioactivation toward 3-MeO-AAB and also 2-aminofluorene and 2-aminoanthracene. The antibody against this P450 completely inhibited mutagenic activation of 3-MeO-AAB of mouse renal microsomes. With immunoblotting, this form was present abundantly in renal microsomes of male mice but not in those of female mice. This P450 was also present in pulmonary microsomes of male and female mice but not in hepatic microsomes. The NH2-terminal amino acid sequence analysis indicated that this form belonged to the CYP4B subfamily. Thus, mouse kidney cDNA library was screened with rat CYP4B1 probe. The cDNA-deduced amino acid sequence of isolated cDNA consisted of 511 amino acids and bore 90, 86, and 84% similarities to rat, rabbit, and human CYP4B1, respectively. The NH2-terminal amino acid sequence of the purified renal P450 and amino acid sequence of BrCN-digested peptides from the purified P450 agreed with the cDNA-deduced amino acid sequence. These results suggest that CYP4B1 is a major form in renal microsomes of male mice and plays a major role in mutagenic activation of 3-MeO-AAB. In extrahepatic tissue, CYP4B1 may contribute to chemical carcinogenesis.

Amino Acid Sequence↗
PubMed1994

Contrary results on mouse hepatitis virus type 3 susceptibility in A/J mouse hepatocytes of phosphatidylserine treatment and of a hypercholesterolaemic diet: no correlation with membrane fluidity levels.

The aim of this study was to examine whether or not membrane fluidity directly influences infection by enveloped viruses, and, more precisely here, the susceptibility of A/J mouse hepatocytes to Mouse Hepatitis Virus type 3 (MHV3). We therefore studied, in parallel, the effects on hepatocyte membrane fluidity and on intracellular viral titre of two treatments, i) a hypercholesterolaemic diet to increase the hepatocyte membrane cholesterol content, ii) direct phosphatidylserine incorporation into hepatocyte membrane. Membrane fluidity was monitored on isolated hepatocytes by fluorescence anisotropy with TMA-DPH, and the viral titre was determined by plaque assay. The results clearly demonstrate that membrane fluidity is not directly involved in viral infection mechanisms.

Animals↗
PubMed1997

Cloning of a mouse smoothened cDNA and expression patterns of hedgehog signalling molecules during chondrogenesis and cartilage differentiation in clonal mouse EC cells, ATDC5.

Hedgehog (hh) family proteins appear to use the conserved targets in their signalling pathway including Patched (Ptc), Smoothened (Smo), and Gli. Although Indian hedgehog (Ihh) plays an important role in endochondral bone formation, the involvement of hh signalling molecules in skeletogenesis is unknown. We cloned a mouse (m) Smo cDNA and studied the expression patterns of Ihh, Ptc, Smo, and Gli mRNAs in mouse chondrogenic EC cells, ATDC5. The deduced amino acid sequence of mSmo consisted of 793 amino acids and was 98 and 93% homologous to the rat (r) Smo and human (h) Smo, respectively. In ATDC5 cells, the expression of Ihh mRNA paralleled that of type X collagen mRNA. Smo, Ptc, and Gli mRNAs were constitutively expressed throughout chondrogenesis and the subsequent cartilage differentiation processes except for the transient decrease in Ptc mRNA at the cellular condensation stage. Our data suggest that hh signalling molecules may be involved in chondrogenesis and cartilage differentiation in ATDC5 cells.

Amino Acid Sequence↗
PubMed1998

Transcriptional activation of the mouse HSP47 gene in mouse osteoblast MC3T3-E1 cells by TGF-beta 1.

HSP47 is a 47-kDa collagen-binding heat shock protein, the expression of which is always correlated with that of collagens in various cell lines. We examined the effects of TGF-beta 1, which is reported to induce the collagen genes, on the expression of HSP47 in mouse osteoblast MC3T3-E1 cells. Treatment of the cells with 5 ng/ml TGF-beta 1 for 24 h increased the level of HSP47 mRNA three-fold. Dose-dependent induction by TGF-beta 1 was observed for both HSP47 mRNA and collagen alpha 1 (I) mRNA, and actinomycin D inhibited this increase of HSP47 mRNA. To elucidate the TGF-beta 1 responsive element(s) in the mouse HSP47 gene, we generated a series of 5'-deletion promoters fused to luciferase reporter constructs. Transient transfection assays showed that TGF-beta 1 induced 4-6 fold the promoter activity of a region approximately -5.5 kbp upstream of the HSP47 gene. Two upstream regions, -3.9 to -2.7 kbp and -280 to -50 bp were shown to be involved in the activation in response to TGF-beta 1 treatment.

3T3 Cells↗
PubMed1998

Long-term postmortem survival of mitochondrial genomes in mouse synaptosomes and their rescue in a mitochondrial DNA-less mouse cell line.

Mitochondrial DNA (mtDNA) transfer was carried out from postmortem mouse tissues to mouse mtDNA-less (rho0) cells to determine how long it takes for autolysis of mtDNA after death and whether mtDNA in postmortem tissues can recover its function in rho0 cells. The results showed that mtDNA was stable in postmitotic tissues stored at 4 degreesC. Moreover, mtDNA in postmortem brain tissues stored for up to 1 month still retained functional properties, causing complete recovery of mitochondrial respiratory function, when it was transferred to rho0 cells. These observations suggest that mtDNA in brain tissue can survive for 1 month after death and can start replication and gene expression in rho0 cells without showing any functional defects. This procedure might be applied to human autopsy brain tissues for examination of the influence of accumulated somatic mutations in mtDNA from aged subjects.

Aging↗
PubMed1998

Characterization of the mouse interleukin-5 promoter in a mouse TH2 T cell clone.

The proximal mouse IL-5 promoter was examined using a mouse TH2 clone stimulated through the T cell receptor using anti-CD3 monoclonal antibody. DNase I protection defined four protein binding regions [IL-5RE-A, -69/-45; -B, (-90/-76); -C, (-154/-130); and -D (-176/-157)]. Stimulation-dependent binding, which was seen in the IL-5RE-B, -D regions and the 5' end of tIL-5RE-A, did not require new protein synthesis inhibitor during cell activation. EMSA using probes targeted to the IL-5RE-B, -C, -D regions demonstrated the multimeric nature of the bound proteins. By transfection analysis using a series of truncated IL-5 promoter-luciferase constructs, IL-5RE-C and -D contributed little to constitutive or inducible activity. The CLE0 site in the IL-5RE-A region contributed to full transcriptional activity but was not sufficient to mediate full activity. Full stimulation-dependent activity required the IL-5RE-B region and/or the GATA site (-70/-60).

Animals↗