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Biomedical subjects

G Brem

Publications and source records attributed to G Brem.

At least 199 records · Page 11Linked to original sources
PubMed1989

Electrofusion parameters for mouse two-cell embryos.

We studied electrofusion of mouse two-cell embryos in order to define parameters which would result in a high yield of fused embryos. Various cell alignment times (from <10 to >60 s) and alternating current percentages (2 to 100%) were examined. The fusion parameters tested were the number of fusion pulses (1-9), pulse length (30-90 mus) and pulse strength (0.50-1.79 kV/cm). Furthermore different combinations of these three parameters were tested. In addition the influence of several embryo culture media on the fusion rates was examined. The results show that the fusion rate of the embryos increases with shorter alignment and higher percentages of the alternating current. The highest fusion rate (95%) was obtained by use of one pulse with a duration of 70 mus and a field strength of 0.60-0.79 kV/cm. The survival rate of the embryos was best if Whitten Medium was used before and after the fusion pulses. The fusion of two-cell stages results in tetraploid embryos which can serve as models for studies in polyploid cells.

Journal Article↗
PubMed1989

Genetic studies on nucleolus organizer regions (NORs) in cattle.

Experimentally produced monozygotic twins, natural opposite sex blood chimeras (freemartins), and several pedigrees were used to evaluate the genetic influences on the nucleolus organizer region (NOR) patterns in cattle. In monozygotic twins, the NOR patterns of both twins are extremely similar. In chimeras, NOR patterns of genetically identical, peripheral blood lymphocytes (PBL) from the two partners resemble each other. In contrast, genetically different PBL (sib organ) differ significantly in the same environment. A high heritability of the individual NOR patterns is also demonstrated in our 23 pedigrees. In conclusion, our data demonstrate that variation in NOR expression is predominantly due to genetic factors.

Animals↗
PubMed1989

Multiple consequences of human growth hormone expression in transgenic mice.

Transgenic mice harbouring growth hormone gene constructs have been produced by DNA microinjection into pronuclei of fertilized oocytes. We examined transgenic mice carrying a mouse metallothionein I-human growth hormone (mMT I-hGH) fusion gene. Here, we present our results concerning gene integration, gene expression, and phenotypical, clinical and pathomorphological alterations found in mice expressing the hGH transgene. Body and organ growth was significantly increased in transgenic mice, whereas fertility was found to be reduced. The life-span was markedly shortened indicating detrimental side-effects of the high levels of circulating hGH. Lesions of kidneys, liver and heart were the predominant pathological findings. Our own results are compared with those obtained by other authors who have investigated mice carrying rat, bovine or ovine growth hormone fusion genes. GH-transgenic mice may serve as a model system to investigate ectopic expression of hormone genes thus circumventing endogenous feedback control mechanisms in complex hormonal cascades.

Animals↗
PubMed1989

[The structure of eukaryotic genes].

The structure of eukaryotic transcription units and already known DNA elements which influence gene expression and eukaryotic gene regulation are described. In recent years molecular genetics approaches have been used to identify and characterize the molecular anatomy of protein-coding eukaryotic genes. Within these genes there are modular sequences, consisting of arrays of short, highly conserved recognition elements that are essential for the initiation of transcription, translation, and tissue-specificity of gene expression. The skillful combination of promoters, enhancers and other regulatory sequences allows the expression of every gene in every organ or tissue of a transgenic animal.

Animals↗
PubMed1989

[Gene transfer in swine by DNA microinjection into zygotes].

The techniques for the production of transgenic pigs are described. Once a gene interesting for animal breeding is found, a gene construct is cloned. The gene construct is a combination of a structure gene and a promoter region which regulates the gene expression of the transgene. The DNA solution is microinjected into the pronuclei of fertilized ova. The injected ova are transferred to synchronised recipient pigs. The animals born must be controlled for the integration of the injected gene construct. In transgenic offspring the expression of the gene, the heredity to the next generation and the biological activity have to be tested. The overall efficiency of a gene transfer programme in pig is about 0.5%. This is lower than in similar projects in mice.

Animals↗
PubMed1989

[Embryo transfer in swine in relation to a gene transfer program].

The production of the recommended embryonic stage for microinjection of foreign DNA into pronuclei needs a precisely timed preparation of donor pigs. For oestrus induction 1250 IU of PMSG is used at a body weight of 60 kg to 90 kg. To induce ovulation 750 IU of HCG are injected 3 days later. After two inseminations eggs are collected by surgical flush of the oviducts and centrifuged for 3 min at 15,000 g for visualization of the pronuclei. After DNA-microinjection the embryos are transferred to 12 h asynchronous recipients. About 50% of the collected eggs could be used for our experiments. The number and quality of the recovered eggs was related to the body weight of the donors. An in vivo culture system in the non-ligated pig oviduct was established in order to measure the effects of the distinct steps in microinjection on embryo survival. The survival rate of untreated zygotes was 52%. After centrifugation the rate was reduced to 38% and after microinjection to 12%. Pregnancy rate after 98 transfers was 37% and an average of 3.9 piglets were born per litter.

Animals↗
PubMed1986

An approach to successful freezing of demi-embryos derived from day-7 bovine embryos.

The developmental capacity of frozen/thawed bisected embryos (n = 33) derived from day-7 bovine embryos was investigated and compared to ordinary embryos after freezing and thawing (n = 28) and to freshly bisected embryos (n = 19). The freezing and thawing protocol was identical for ordinary and demi-embryos. The percentage of intact embryos classified as excellent, good, or poor after thawing was 92.9 and 96.3% for ordinary and demi-embryos, respectively. Pregnancy rates of 53.8 (8 15 ), 46.2 (6 13 ), and 47.5% (9 19 ) were obtained when frozen/thawed ordinary embryos and frozen/thawed demi-embryos classified as excellent or good and sealed with an additional zona pellucida from hatched pig blastocysts or freshly bisected embryos were transferred. One pair of identical twins resulted from the transfer of frozen/thawed demi-embryos sealed with an additional zona pellucida. Transfer of four frozen/thawed demi-embryos without an additional zona pellucida led to one pregnancy. In contrast, demi-embryos derived from frozen/thawed ordinary embryos (n = 8) as well as frozen/thawed demi-embryos classified as poor (n = 6) did not result in any pregnancies although two halves were transferred per recipient. It is concluded that sealing the punctured zona pellucida improves the developmental capacity of frozen/thawed demi-embryos derived from day-7 bovine embryos, and freezing demi-embryos is more efficient compared to the splitting of frozen/thawed ordinary embryos.

Journal Article↗
PubMed1986

[Lipid storage disease similar to Wolman's disease in humans in the fox terrier].

In three young fox terriers a lipid storage disease is reported. This thesaurosis is almost identical to Wolman's disease in children. Clinically and pathologically hepatosplenomegaly is the most striking feature. Morphologically the disease is characterized by extensive lipid deposition mainly in liver, spleen, lymphnodes, intestinal mucosa, and bone marrow. Circular corneal lipid deposit (Arcus lipoides corneae) is of diagnostic significance. Some of the material has the typical structure of cholesterol crystals. Under polarized light the deposits consist of birefringent and non-birefringent lipids. All three dogs with lipid storage disease have one common ancestor. In addition their pedigrees reveal close relationship amongst the nearer forefathers and the affected animals themselves. From analogy with human Wolman's disease it is concluded, that in the Foxterrier, too, this lipid storage disease is caused by an inheritable deficiency of acid esterase. The mode of inheritance of this inborn lysosomal disease is probably autosomal recessive.

Animals↗
PubMed1985

[Production of chimeras by embryo microsurgery and their possible application in cattle breeding].

An important contribution to genetic and embryonic engineering in mammalian embryos is the production of chimeric mammals. Chimeras can be produced by aggregating the blastomeres of two different embryos to make a single individual. A method is described to produce bovine chimeras by aggregating non-surgically collected embryos of different breeds and transferring the aggregated embryos nonsurgically to synchronised recipients. An overt chimeric bull calf (Holstein-Friesian and Brown-Swiss) was born on March 31st, 1984. Chimeras have many uses in research, but one practical use is to rescue defective embryos that would not develop to term on their own. Such embryos can be aggregated with normal blastomeres to produce a chimera. Some of the initially defective cells can even become part of the germ line in the chimera and give rise to perfectly normal eggs or sperm. Two models for using this technique in cattle research are described. The lethal disorder arachnomelie has a rather high frequency in some German Brown-Swiss populations. Chimeric calves developing from embryos descending of carrier matings could help to find out the genetic and developmental background of this disorder. Chimeras produced of transgenic teratocarcinoma cell lines and normal embryos offer one possibility to produce transgenic cattle. The advantages and disadvantages of these possibilities are discussed.

Animals↗
PubMed1984

Chimerism in cattle through microsurgical aggregation of morulae.

A cattle chimera was produced by combining four halves of two parent embryos of different breeds (Brown-Swiss x Braunvieh plus Holstein-Friesian x Holstein-Friesian) in one zona pellucida. Parent embryos in the 32-cell morula stage were recovered non-surgically, were bisected, and the combined four halves were transferred non-surgically to recipient heifers. Chimerism of coat colour was used as evidence. Combining of only two half embryos from different parents resulted in five pregnancies carried to term but none of the calves born was a chimera.

Journal Article↗
PubMedDate not supplied

Transfection of epithelial cells is enhanced by combined treatment with mannitol and polyethyleneglycol.

BACKGROUND: Gene transfer efficiency drops significantly when polarized mammary epithelial cells are transfected instead of actively growing cells. However, fully differentiated cells are the targets for gene transfer in many in vivo applications. Therefore, a simple and effective method for the transfection of polarized mammary epithelial cells in confluent monolayers was developed. METHODS: Reporter gene plasmids were complexed with polyethylenimine with an average molecular weight of 25 kDa (PEI 25), or other agents, to transfect confluent monolayers of ovine mammary epithelial cells (OMEC II) or human carcinoma cells (CaCo-2) in vitro. The improved technique included pretreatment of the cells with a hyperosmotic mannitol solution (7%) which caused a loosening of the tight contacts between the cells. Alternatively, the mannitol shock could be replaced by a short treatment with trypsin or EDTA. In addition to the pretreatment, 12.5% polyethyleneglycol with an average molecular weight of 8000 kDa (PEG 8000) was included in the transfection mixture containing the DNA complexes. RESULTS: The combined application of mannitol and PEG resulted in a very reliable 5- to 30-fold increase in reporter gene expression in OMEC II and CaCo-2 cells, but not K562 cells (an example of another cell type). The improved technique can also be combined with other polymer-based transfection agents. The transfection rate was enhanced for confluent monolayer cells with fully developed epithelial polarity but also for subconfluent, growing epithelial cell cultures. CONCLUSIONS: A novel transfection protocol for epithelial cells is presented. The combined treatment of cells with mannitol and polyethyleneglycol results in substantial enhancement of in vitro transfection of epithelial cell lines.

Animals↗
PubMedDate not supplied

[A composite polyaniline-containing silica sorbent for DNA isolation].

A composite sorbent based on porous glass beads modified with thin polyaniline coating was prepared by precipitating aniline polymerization in the presence of carrier particles. It was shown that the modification ensures the uniform coating of the inner surface of the carrier pores with the polymer layer approximately 70 A thick. It was shown that the resulting material retains the initial porosity of the carrier and is selective in the separation of nucleic acids and proteins. The polyaniline-coated sorbents were shown to be efficient for both the preparative DNA isolation from bacterial lysates and for analytical purposes, in particular, for studying DNA fragmentation during apoptosis proceeding under UV irradiation of cell lysates of colon carcinoma. The morphological and chromatographic characteristics of the new sorbent were demonstrated to be similar to those of the polyfluorobutadiene sorbent.

Aniline Compounds↗
PubMedDate not supplied

Cytogenetic, ras, and p53: studies in cases of canine neoplasms (hemangiopericytoma, mastocytoma, histiocytoma, chloroma).

Four case reports of mesenchymal neoplasms showing chromosomal abnormalities are presented. In a case of hemangiopericytoma trisomy 2 and centric fusion 19;21 were present. In a mastocytoma a deleted chromosome 35 was seen. A homogeneously staining region (HSR) on chromosome 1 was detected in a histiocytoma. Trisomy 5 and monosomy 31 were observed in a case of granulocytic sarcoma (chloroma). The lack of mutations in exons 1 and 2 of oncogenes N-ras, K-ras, and H-ras and exons 5, 6, 7, and 8 of tumor suppressor gene p53 in these four patients and in a larger series of investigated dogs (25 hemangiopericytomas, 12 mastocytomas, and 8 histiocytomas) is highlighted.

Animals↗