Search PubMed⌕ Search

Biomedical subjects

G Brem

Publications and source records attributed to G Brem.

At least 163 records · Page 9Linked to original sources
PubMed1993

Influence of beta-carotene on fertility in rabbits when using embryo transfer programs.

The effect of beta-carotene on reproduction traits in rabbits was studied in 509 (superovulated and normally ovulated) donors and 239 recipients by using embryo/gene transfer performed at 2 different locations. All of the bucks and the half of the females were fed a diet supplemented with 40 mg synthetic beta-carotene (Rovimix((R)))/kg feed. Embryos at the pronucleus stage were collected 19 to 21 hours after induction of ovulation with human chorionic gonadotropin (hCG); they were then microinjected into the male pronucleus and transferred to synchronized recipients. Data were obtained from the time when the donors and recipients were caged, until the pups resulting from the embryo transfers were weaned. Supplemented beta-carotene did not affect most of the 30 traits that were analyzed. However superovulated donors in Project 2 that received the beta-carotene enriched diet had a 14% lighter ovary weight (P<0.05) and less than half of the oocytes were unfertilized (P<0.05). In Project 1 (beta-carotene group) there was a greater number of pups born (36%, P<0.05) and more of these pups were born alive (53%, P<0.05).

Journal Article↗
PubMed1993

Heart-specific targeting of firefly luciferase by the myosin light chain-2 promoter and developmental regulation in transgenic mice.

Based on hybridization studies indicating constitutive expression levels of the endogenous myosin light chain-2 (MLC-2) gene in embryonic, fetal, and adult myocardium, a model system for selective targeting of genes to the heart of transgenic mice has been developed. A 2.1-kb DNA fragment of the 5' flanking region of the rat cardiac MLC-2 gene was fused to the firefly luciferase reporter gene and introduced into fertilized mouse oocytes. In four independent transgenic mouse lines, the expression of the MLC-2-luciferase fusion gene was found exclusively in heart muscle. In contrast to the endogenous MLC-2 gene, no luciferase activity was detectable in slow-twitch skeletal muscle or any other tissue of transgenic mice. This result suggests that the 2.1-kb DNA fragment of the 5' flanking region of the cardiac MLC-2 gene contains the regulatory elements required for selective gene expression in cardiac myocytes in vivo. In contrast to the endogenous steady-state MLC-2 expression during development, transgenic luciferase activity was 10-fold higher during embryogenesis, when formation of the ventricular loop and septum takes place. The enhanced luciferase activity in early heart development may suggest a growth-dependent control mechanism, involving either transcriptional or posttranscriptional regulation. In conclusion, this model system with the 2.1-kb ventricle-specific MLC-2 promoter sequence should facilitate the overexpression of gene products in the developing and mature heart muscle and further elucidate molecular mechanisms of myocardial diseases such as cardiomyopathies.

Animals↗
PubMed1993

Laparoscopic embryo transfer in rabbits.

A technique for endoscopic transfer of one-cell, one-two-cell, two-cell or morula stage embryos to the Fallopian tubes of rabbits was developed in two phases. Phase I of the experiments involved the transfer of 30-65 embryos to each of nine recipients and resulted in pregnancies in all animals. After 12 days six rabbits were killed and they showed an implantation rate of 27%. The remaining three rabbits continued pregnancy until birth. The embryo survival rate was 26%. In a second phase, 10-20 embryos were transferred to each of the 22 recipient animals, 19 of which went on to give birth (embryo survival rate in the animals that became pregnant was 47%).

Animals↗
PubMed1993

Sub-retroviral particles as gene transfer vectors that by-pass retrovirus-receptor interaction restrictions.

Retroviral infection initiates with the binding of the virus envelope protein to cell membrane receptors. However, it is unclear whether this interaction is necessary for subsequent events required to establish infection. To address this problem subretroviral particles containing a retroviral vector genome carrying a beta-galactosidase (beta-gal) gene have been microinjected into cells. Here we show that such cells become infected as judged by expression of beta-gal activity, suggesting that virus-receptor interaction is not required for infection. Such subretroviral particles lacking an envelope may be useful both for safer gene transfer procedures and gene transfer to cells that do not express the cognate virus receptor.

3T3 Cells↗
PubMed1992

Transgenic pigs carrying cDNA copies encoding the murine Mx1 protein which confers resistance to influenza virus infection.

An important aspect of gene transfer into farm animals is the improvement of disease resistance. The mouse Mx1 protein is known to be sufficient to confer resistance to influenza viruses. Gene constructs containing the mouse Mx1 cDNA controlled by the human metallothionein IIA promoter (hMTIIA::Mx), the SV40 early enhancer/promoter region (SV40::Mx) and the mouse Mx1 promoter (mMx::Mx) were transferred into pigs. The results of the gene transfer experiments with the hMTIIA::Mx and the SV40::Mx constructs indicate that the permanent high-level synthesis of Mx1 might be deleterious to the organism: the gene transfer efficiency was surprisingly low, and all transgenic piglets born had rearrangements in their transgene copies that abolished protein synthesis. The use of the interferon (IFN)- and virus-inducible mMx::Mx construct resulted in normal gene transfer efficiency. Two transgenic pig lines could be established which expressed IFN-inducible mouse Mx1 mRNA. Extensive protein analysis did not detect mouse Mx1 in IFN-treated transgenic animals.

Animals↗
PubMed1992

Myosin light chain-2 luciferase transgenic mice reveal distinct regulatory programs for cardiac and skeletal muscle-specific expression of a single contractile protein gene.

To examine the relationship between the cardiac and skeletal muscle gene programs, the current study employs the regulatory (phosphorylatable) myosin light chain (MLC-2) as a model system. Northern blotting, primer extension, and RNase protection studies documented the high level expression of the cardiac MLC-2 mRNA in both mouse cardiac and slow skeletal muscle (soleus). Transgenic mouse lines harboring a 2100- or a 250-base pair rat cardiac MLC-2 promoter/luciferase fusion gene were generated, demonstrating high levels of luciferase activity in cardiac muscle, and only background luminescence in slow skeletal muscle and non-muscle tissues. As assessed by in situ hybridization, immunofluorescence, and luminescence assays of luciferase reporter activity in various regions of the heart, both the endogenous MLC-2 gene and the MLC-2 luciferase fusion gene were expressed exclusively in the ventricular compartment, with expression in the atrium at background levels. Point mutations within the conserved regulatory sites HF-1a and HF-1b significantly cripple ventricular muscle specificity, while mutation of the single E-box site was without effect, suggesting that ventricular muscle-specific expression occurs through an E-box-independent pathway. This study provides direct evidence that the cis regulatory sequences in the cardiac/slow twitch MLC-2 gene which confer cardiac and skeletal muscle-specific expression can be clearly segregated, suggesting that distinct regulatory programs may have evolved to control the tissue-specific expression of this single contractile protein gene in cardiac and skeletal muscle.

Amino Acid Sequence↗
PubMed1992

Genomic organization and analysis of the 5' end of the porcine ryanodine receptor gene (ryr1).

In this study we describe the isolation of genomic clones of the 5' region of the porcine ryanodine receptor gene, a candidate for malignant hyperthermia in pigs and humans. The recombinants were isolated from a porcine liver, genomic DNA library in phage EMBL3A after screening with PCR amplified DNA fragments. The exon/intron structure of the ryanodine receptor gene was determined by DNA sequencing. Based on the sequence data it was possible to develop a simple test for the detection of malignant hyperthermia susceptible and normal pigs.

Animals↗
PubMed1992

[Embryo cloning in domestic animals].

The cloning of livestock is performed by the nuclear transfer of early embryonic stages into prepared oocytes in order to obtain a high number of genetic identical animals. As the most important technical steps there are maturation and enucleation of the oocytes, isolation of single blastomeres or karyoplasts of the donor embryo, transfer of the nucleus-containing membrane vesicle under the zona pellucida of the recipient cell, and fusion of the recipient cell and blastomere or karyoplast. Until now, the largest clone which is known exists of seven bulls. The efficiencies of the particular methodical steps have to be improved. More knowledge of the activation of oocytes, nucleus differentiation and availability of determined cell cycle-stages of mitosis is required. The combination of embryo cloning, cryopreservation of embryos and non-surgical embryo transfer is required for basic research and animal breeding.

Animals↗
PubMed1992

Molecular cloning of porcine Mx cDNAs: new members of a family of interferon-inducible proteins with homology to GTP-binding proteins.

Porcine cells treated with interferon (IFN) or double-stranded RNA synthesize two proteins that exhibit high homology of the amino acid sequence to mouse Mx1 protein involved in selective resistance to influenza virus. A full-length cDNA clone (poMx1) encoding the porcine Mx1 protein was isolated and sequenced. It contained an open reading frame of 663 amino acids. The predicted molecular weight of 75.6 kD is in good agreement with the apparent molecular mass of the two immunoprecipitable proteins of 76 kD and 73 kD determined by SDS polyacrylamide gel electrophoresis. A second cDNA (poMx2) was characterized which was incomplete in the 5' region. A comparison of all known Mx proteins revealed an average homology of 67.5%. The porcine Mx1 polypeptide is most closely related to human MxA (p78), murine Mx2, rat Mx2, and rat Mx3 proteins. The amino-terminal halves of all Mx proteins are highly conserved and possess three consensus elements in proper spacing, characteristic of GTP-binding domains. The Mx family shows in their amino termini striking homology to previously characterized Mx-related proteins playing roles in the intracellular vectorial transport of proteins--the products of the yeast Vps1 locus and the dynamins.

Amino Acid Sequence↗
PubMed1992

The GH-transgenic mouse as an experimental model for growth research: clinical and pathological studies.

The objectives and the methodology of mammalian gene transfer are discussed and findings in growth hormone (GH) transgenic mice are reported to illustrate the potential offered by genetically designed animal models for investigations in various areas of biomedical research. Transgenic mice expressing hybrid genes composed of either human or bovine GH coding sequences fused to the mouse metallothionein I promoter show high serum levels of heterologous GH, increased growth rates and final adult size, decreased life expectancy and a variety of pathological changes.

Animals↗
PubMed1992

Pregnancy rates and births after unilateral or bilateral transfer of bovine embryos produced in vitro.

Late morulae and blastocysts produced in vitro were nonsurgically transferred to heifers by unilateral (n = 184) or bilateral (n = 94) transfer. Of the recipients, 58% had serum progesterone values greater than 1.4 ng ml-1 on day 21 and rectal palpation on day 35 showed that 50% (138 of 278) were pregnant. The embryonic mortality rate between days 21 and 35 was estimated to be about 14% and between days 36 and 90 to be about 12%. Of the animals, 8% aborted between days 91 and 250 of pregnancy. No difference was observed in pregnancy rates between unilateral transfer of one (47%) or two embryos (49%) and bilateral transfer (53%), or in the twinning rate between bilateral transfer (42%) and unilateral transfer of two embryos (33%). The pregnancy rate was 54% with embryos evaluated as morphologically excellent or good, 51% with fair embryos and 26% with poor ones. A higher pregnancy rate (60%) was obtained after embryo transfer when the synchrony between recipient and embryo was -1 day.

Abortion, Veterinary↗
PubMed1991

Disease resistance in farm animals.

Genetic variations in disease resistance of farm animals can be observed at all levels of defence against infectious agents. In most cases susceptibility to infections has polygenic origins. In domestic animals only a few instances of a single genetic locus responsible for disease resistance are known. A well-examined example is the Mx1 gene product of certain mice strains conferring selective resistance to influenza virus infections. Attempts to improve disease resistance by gene transfer of different gene constructs into farm animals include the use of monoclonal antibody gene constructs, transgenes consisting of antisense RNA genes directed against viruses and Mx1 cDNA containing transgenes.

Animals↗
PubMed1991

Genes encoding a mouse monoclonal antibody are expressed in transgenic mice, rabbits and pigs.

To study the expression pattern of immunoglobulin-encoding genes in transgenic animals, we have introduced the genes for the light and heavy chain of a mouse monoclonal antibody (mAb) into the germ-line of mice (control), rabbits and pigs. The transgenes were detected in the mouse lines, two rabbit lines and pigs. Titers of 100-200 micrograms mAb/ml (rabbits) and up to 1000 micrograms mAb/ml (pig) were measured in the sera of the transgenic animals. Isoelectric focusing experiments with serum followed by immunofixation revealed that in the transgenic pig only a minority of the bands were identical to those of the purified mouse mAb. In transgenic rabbits we found no coincidence of bands at all. The results can be explained by assuming tissue- and cell-type-specific glycosylation, modification and possible heterologous chain associations. Expression of Ab in the serum of animals could help to protect against diseases (e.g., influenza in pigs).

Animals↗
PubMed1991

Human VNTR sequences in porcine HTF-islands.

Mapping of complex genomes has been influenced substantially by the isolation of locus-specific, but repetitive DNA elements known as VNTRs. Since a high GC-content is characteristic of most of these elements one might expect them to be clustered at least partially in CpG-islands. To address this question we have constructed a porcine liver DNA NotI-linkage library in pUC18 using isolated HTF-islands. HpaII tiny fragments ranging from less than 100 bp to 1 kb in length were randomly selected and analysed by sequencing. As expected from the source of DNA the clones were rich in CpG-, C- and G-content. Several clones exhibiting a repetitive (GGC)n-motif and sequences originally present in human VNTR-markers were shown to detect DNA fingerprints in different species (Homo sapiens, Sus scrofa domestica, Gallus domesticus, Glycine max, Saccharomyces cerevisiae). A subset of clones was used for hybridization experiments with porcine DNA and were shown to detect single-copy linkage fragments.

Animals↗
PubMed1991

Developmental and tissue-specific expression of the Q5k gene.

Expression of the Q5k gene was examined by northern blot analysis and polymerase chain reaction (PCR) in the AKR mouse and various cell lines, each of the H-2k haplotype. Our results show that Q5k mRNA is present during the whole postimplantational development of the AKR embryo/fetus (gestation day 6 to 15). In the juvenile mouse (week 2 to 4) transcription of the Q5k gene persisted in all organs examined. In contrast, in the adult animal expression of the Q5k gene was limited to the thymus and uterus of the pregnant mouse. Upon malignant transformation, the amount of Q5k-specific mRNA increased dramatically in thymus and could also be observed in the spleen of thymoma bearing animals. Expression of the Q5k gene was also detectable in several transformed mouse cell lines. Mitogen stimulation or treatment with cytokines induced Q5k expression in primary spleen cell cultures. A possible explanation for the tissue-restricted expression in the adult AKR mouse is discussed.

Aging↗
PubMed1991

Accelerated growth and visceral lesions in transgenic mice expressing foreign genes of the growth hormone family: an overview.

Effects of growth hormone (GH) overproduction were studied in transgenic mice expressing murine metallothionein I-GH fusion genes. The most obvious consequence was the acceleration of growth, which led to substantial increases in body weight of up to more than twice that seen in controls. Growth of the internal organs was stimulated, with hepatomegaly and nephromegaly as the most prominent features. GH transgene expression was also reflected in increased skeletal growth which affected various bones to different extents. The mean life-span of human GH transgenic mice with serum levels of hGH ranging from 3 x 10(3) to 9 x 10(5) ng/ml was drastically reduced at 160 days in both sexes. Severe renal lesions were the primary cause of the decrease in life expectancy and were characterized by marked nephron atrophy, obsolescence of numerous glomeruli, and a massive cystic dilation of the tubules. Initial changes involved the glomeruli, which showed significant enlargement and sclerotic lesions. The liver exhibited a pronounced hepatocellularmegaly and progressive degenerative as well as hyperplastic changes. One-third of the hGH transgenic animals displayed myocardial fibrosis. Hepatocellular carcinoma was found in bovine GH transgenic mice older than 12 months. Our observations are compared with results of other investigators.

Animals↗