DNA microsatellite analysis for parentage control in Austrian pigs.

We present an efficient parentage control for pigs based on ten polymorphic microsatellite markers analyzed in a single PCR reaction. Assuming one known parent ("paternity control"), combined exclusion probabilities (CEPs) ranged from 99.18% (Landrace), 99.74% (Piétrain) to 99.76% (Large White) for the most important Austrian breeds. Assuming a known parent-pair ("parentage control", e.g. a substituted offspring), the CEP of the 10-plex PCR increased to 99.97% (Landrace) and 99.99% (Piétrain and Large White). We developed an additional standby battery of 5 markers, which might be applied in those cases, where the CEP of the 10-plex PCR is not sufficient. Therefore an automated, cost and time reduced genotype analysis for pigs is available.     

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 70 Å 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.     

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.     

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 µg/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. Copyright © 2002 John Wiley & Sons, Ltd.

     

Inbreeding,microsatellite heterozygosity,and morphological traits in Lipizzan horses

While the negative effects of inbreeding and reduced heterozygosity on fecundity and survival are well established, only a few investigations have been carried out concerning their influence on morphological traits. This topic is of particular interest for a small and closed population such as the Lipizzan horse. Thus, 27 morphological traits were measured in 360 Lipizzan mares and were regressed on the individual inbreeding coefficients, as well as on the individual heterozygosity and mean squared distances (mean d(2)) between microsatellite alleles within an individual. Both individual heterozygosity and mean d(2) were based on 17 microsatellite loci dispersed over 14 chromosomes. The results obtained by multivariate analysis reveal significant effects of stud (P <.0001), age at measurement (P <.0001), and mean d(2) (P =.0143). In univariate analyses, significant associations were obtained between length of pastern-hindlimbs and inbreeding coefficient (P <.01), length of cannons-hindlimb and mean d(2) (P <.01), and length of neck and mean d(2) (P <.001). After adjustment of single-test P values for multiple tests (Hochberg's step-up Bonferroni method), only the association of the length of neck and mean d(2) remained significant (P =.0213). Thus, no overall large effects of inbreeding, microsatellite heterozygosity, and mean d(2) on morphological traits were observed in the Lipizzan horse.     

Efficient transgenesis in farm animals by lentiviral vectors

Microinjection of DNA is now the most widespread method for generating transgenic animals, but transgenesis rates achieved this way in higher mammals are extremely low. To address this longstanding problem, we used lentiviral vectors carrying a ubiquitously active promoter (phosphoglycerate kinase, LV-PGK) to deliver transgenes to porcine embryos. Of the 46 piglets born, 32 (70%) carried the transgene DNA and 30 (94%) of these pigs expressed the transgene (green fluorescent protein, GFP). Direct fluorescence imaging and immunohistochemistry showed that GFP was expressed in all tissues of LV-PGK transgenic pigs, including germ cells. Importantly, the transgene was transmitted through the germ-line. Tissue-specific transgene expression was achieved by infecting porcine embryos with lentiviral vectors containing the human keratin K14 promoter (LV-K14). LV-K14 transgenic animals expressed GFP specifically in basal keratinocytes of the skin. Finally, infection of bovine oocytes after and before in vitro fertilization with LV-PGK resulted in transgene expression in 45% and 92% of the infected embryos, respectively.     

Microsatellite analysis reveals substantial genetic differentiation between contemporary New World and Old World Holstein Friesian populations

Genotypic data from 39 microsatellite loci typed in 211 animals were used to assess the genetic differentiation between Old World and New World Holstein Friesian cattle populations. Gene diversities were similar in all five Holstein Friesian populations surveyed, ranging from 0.43 to 0.48. A tree of individuals based on the proportion of shared alleles indicated a clear distinction between Old World and New World Holstein Friesian populations. Similarly, genetic differentiation between populations, as measured by FST, was highly significant. Using the split decomposition method, we were able to visualize the significant introgression of New World Holstein Friesian into European Holstein Friesian populations.     

Assay for drug discovery: Synthesis and testing of nitrocefin analogues for use as β-lactamase substrates

We report on the synthesis of three nitrocefin analogues and their evaluation as substrates for the detection of β-lactamase activity. These compounds are hydrolyzed by all four Ambler classes of β-lactamases. Kinetic parameters were determined with eight different β-lactamases, including VIM-2, NDM-1, KPC-2, and SPM-1. The compounds do not inhibit the growth of clinically important antibiotic-resistant gram-negative bacteria in vitro. These chromogenic compounds have a distinct absorbance spectrum and turn purple when hydrolyzed by β-lactamases. One of these compounds, UW154, is easier to synthesize from commercial starting materials than nitrocefin and should be significantly less expensive to produce.     

Metformin Inhibits Growth of Human Glioblastoma Cells and Enhances Therapeutic Response

High-grade gliomas, glioblastomas (GB), are refractory to conventional treatment combining surgery, chemotherapy, mainly temozolomide, and radiotherapy. This highlights an urgent need to develop novel therapies and increase the efficacy of radio/chemotherapy for these very aggressive and malignant brain tumors. Recently, tumor metabolism became an interesting potential therapeutic target in various cancers. Accordingly, combining drugs targeting cell metabolism with appropriate chemotherapeutic agents or radiotherapy has become attractive. In light of these perspectives, we were particularly interested in the anti-cancer properties of a biguanide molecule used for type 2 diabetes treatment, metformin. In our present work, we demonstrate that metformin decreases mitochondrial-dependent ATP production and oxygen consumption and increases lactate and glycolytic ATP production. We show that metformin induces decreased proliferation, cell cycle arrest, autophagy, apoptosis and cell death in vitro with a concomitant activation of AMPK, Redd1 and inhibition of the mTOR pathway. Cell sensitivity to metformin also depends on the genetic and mutational backgrounds of the different GB cells used in this study, particularly their PTEN status. Interestingly, knockdown of AMPK and Redd1 with siRNA partially, but incompletely, abrogates the induction of apoptosis by metformin suggesting both AMPK/Redd1-dependent and –independent effects. However, the primary determinant of the effect of metformin on cell growth is the genetic and mutational backgrounds of the glioma cells. We further demonstrate that metformin treatment in combination with temozolomide and/or irradiation induces a synergistic anti-tumoral response in glioma cell lines. Xenografts performed in nude mice demonstrate in vivo that metformin delays tumor growth. As current treatments for GB commonly fail to cure, the need for more effective therapeutic options is overwhelming. Based on these results, metformin could represent a potential enhancer of the cytotoxic effects of temozolomide and/or radiotherapy.