Biomedical subjects

Karl Schellander

Publications and source records attributed to Karl Schellander.

24 records · Page 2Linked to original sources

PubMed2003

Association of the porcine C3 gene with haemolytic complement activity in the pig.

The complement component C3 plays an essential role in the activated complement system, which is involved in phagocytosis, inflammation and immunoregulation to destroy infectious microorganisms. The C3 molecule has more implications in the general defence mechanisms. In this study, the porcine C3 cDNA sequences including 5'- and 3'- flanking regions were determined and the polymorphisms in this gene were identified to carry out an association analysis between C3 and complement activity traits. Porcine C3 gene has high homology with human C3. Five single nucleotide polymorphisms (SNPs) and one microsatellite were detected in the porcine C3 gene. Haemolytic complement activity of alternative and classical pathways (ACH, CCP) was measured in 416 F2 animals of a crossbred of Duroc x Berlin Miniature Pig, which were immunized with Mycoplasma, Aujeszky and PRRS vaccines. C3 markers were found to be significantly associated (P <0.05) with both ACP and CCP. Animals with the more frequent haplotype present in Duroc and other commercial breeds exhibit higher ACP and CCP levels than the animals with haplotype specific to some Berlin Miniature Pigs. The association of C3 with complement activity reinforces the importance of C3 as a candidate gene for natural resistance to microorganisms.

Animals↗

PubMed2003

[Some factors affecting in vitro development of porcine embryo reconstitution from somatic cells nuclear transfer].

In this paper, a study on reconstitution of porcine oocytes by using nuclear transfer with cumulus cells(CC) and fibroblast cells(FC) was carried out. Reconstituted oocytes which were the fusion with CC and showed a cleavage rate of 56.7%, developed to morula(11.7%) and blastocysts(6.7%) phases which were higher than those derived from the fusion with FC(p < 0.05). The results of this study also involved the effects of oocyte collection method and maturational age of recipient oocytes during the in vitro development of nuclear-transfer embryos which were reconstructed with cultured cumulus cells. The cumulus cells synchronized in G0/G1 phases through serum-starvation culture, were transferred into enucleated oocytes which were collected by aspiration or dissection method and cultured for 33 or 44 h. Reconstituted embryos were activated with a combination of calcium ionophore A23187 or electric pulsation and 6-DMAP, and cultured for 6 days. As for the oocytes collection methods, activation treatment in the presence of cytochalasin B did not affect the developmental rate of embryos reconstituted with 44-h-mature recipients. However, the development rate of reconstituted embryos with 33-h-mature recipients was significantly higher(p < 0.05) by activation with the combination of electric pulsation and 6-DMAP. These results suggest that reconstituted porcine embryos derived from cultured cumulus cells can develop to the blastocyst stage and that the development of the former could be improved by reconstruction with young oocyte cytoplast after the activation with the combination of electric pulsation and 6-DMAP.

Adenine↗

PubMed2002

A source for expression profiling in single preimplantation bovine embryos.

Our knowledge of the genes active during normal preimplantation development in cattle is limited, despite the importance for further improvement of fertility and applicability of biotechniques, like in vitro production and embryo transfer. We report on the construction of cDNA libraries as a source for expression profiling in oocytes and single preimplantation cattle embryos. cDNAs were prepared from two unfertilized oocytes, single two-cell, four-cell and eight-cell, morula, and blastocyst stage embryos, respectively. The oocytes, eight-cell, morula, and blastocyst stage embryo-derived cDNAs were ligated to a lambda-based expression vector and these have complexities of 8 x 10(5), 5 x 10(5), 1 x 10(6) and 2 x 10(6) independent clones, respectively. A total of 48 clones were picked and sequenced, 62.5% (30/48) of the sequence were homologous to known transcripts from human and mouse, 18.75% (9/48) to expressed sequence tags (ESTs) of human and mouse origin. Novel sequences were detected at a frequency of 14.58% (7/48). PCR analyses of the embryonic libraries for specific genes revealed transcripts for genes including housekeeping genes (GAPDH and beta-actin), developmental genes (OCT-4, IGF-I receptor and homeodomain sequences) and genes coding for metabolic and protective enzymes (manganese superoxide dismutase, glutamine synthetase, flavin-containing mono-oxygenase, glutamate dehydrogenase, alpha-2-macroglobulin). These cDNA libraries are a valuable resource for the isolation of clones representing genes active at these early developmental stages. The ability to construct cDNA expression libraries from only a few cells will allow gene expression analyses from embryo biopsies and embryos derived by nuclear transfer procedures.

Animals↗

PubMed2002

Stage-specific expressed sequence tags obtained during preimplantation bovine development by differential display RT-PCR and suppression subtractive hybridization.

Differential display RT-PCR (DDRT-PCR) and suppression subtractive hybridization (SSH) were applied in order to detect preimplantation stage-specific expressed sequence tags (ESTs) of bovine embryos. Seventeen ESTs were detected from the differential display RT-PCR approach. All clones but two showed homology to genes or ESTs known in human, cattle or other species. One of the clones similar to H. sapiens mRNA for KIAA1764 protein was used exemplarily to quantify the transcripts by real-time PCR. The result of quantitative differential screening was found to be in agreement with DDRT-PCR banding patterns. In the second approach, a blastocyst-stage enriched cDNA library was constructed using SSH of blastocyst versus morula transcripts. The 71 clones that were analysed represent 33 distinct loci including candidate genes for the regulative processes during differentiation of inner cell mass (ICM) and trophoblast cells and the initial phase of embryo implantation, such as galectin-3 and fibronectin. As revealed by real-time PCR, the mRNA level of galectin-3 was three times higher in the blastocyst stage than in the morula stage. DDRT-PCR and SSH are both powerful tools for the identification of stage-specific expressed gene in preimplantation bovine embryos. Real-time PCR allows to test and confirm the outcome and to add quantitative data of selected transcripts of interest.

Animals↗

PubMed2002

Detection of quantitative trait loci for carcass traits in the pig by using AFLP.

For evaluation of the suitability of Amplified Fragment Length Polymorphism (AFLP) for detection of quantitative trait loci in farm animals, a combination of AFLP and selective genotyping has been applied as a rapid screening method for marker-QTL associations. Focusing on loci affecting eye muscle area, six extreme discordant sib pairs were selected from a Duroc x Berlin Miniature Pig F2 experimental cross and examined by using 48 AFLP primer combinations. Two prominent AFLP markers were converted into simple codominant PCR markers ( STS-Bo1 and STS-Bo3) and assigned to Sscr4 by physical and linkage mapping. Single marker analysis indicated association of the STS markers with a putative QTL influencing eye muscle area. Interval mapping confirmed the presence of a significant QTL for eye muscle area (Pgenomewide < 0.01) on the Sscr4, with STS-Bo1 being the closer marker. At the same location, significant effects (Pgenomewide < 0.01) on carcass length and backfat thickness were also detected. Our results demonstrate the capability of the combination of AFLP analysis and selective genotyping as a method for detection of genome regions containing QTL in livestock.

Animals↗

PubMedDate not supplied

Somatic gene transfer into the lactating ovine mammary gland.

BACKGROUND: Somatic gene therapy requires safe and efficient techniques for the gene transfer procedure. The ovine mammary gland is described as a model system for the evaluation of somatic gene transfer methods. METHODS: Different gene delivery formulations were retrogradely injected into the mammary gland of lactating sheep. The efficiency of the gene transfer was subsequently measured by the detection of the secreted transgene products in the milk. To counteract the milk flow in the lactating gland caused by the permanent milk production, a newly developed pretreatment of the mammary gland with hyperosmotic solutions was applied. In addition, in vivo electroporation of DNA into the mammary gland is described. RESULTS: Gene transfer using naked DNA or simple complexes of DNA with polycations did not result in traceable amounts of reporter gene products. However, utilizing the complex cationic lipid DOSPER, a peak expression of about 400 ng/ml was observed 6 days after transfection. Maximum expression rates of more than 1 microg/ml were obtained by combining hyperosmotic pretreatment and receptor-mediated gene transfer. For the in vivo electroporation, the proof of principle for this technique in the mammary gland is reported. CONCLUSIONS: The ovine mammary gland turned out to be a very well suited as a model system for evaluation and optimization of various gene transfer protocols.

Animals↗