The absence of detectable fetal microchimerism in nontransgenic goats (Capra aegagrus hircus) bearing transgenic offspring.

Regulations for the disposal of genetically engineered animals are strict due to concern for their inappropriate introduction into the food chain, and of the possible public health and environmental impacts of these organisms. Nontransgenic animals that give birth to transgenic offspring are treated as if they are transgenic due to concern of fetal cells crossing the placental barrier and residing in the mother (fetal-maternal microchimerism). Determining whether or not fetal-fetal or fetal-maternal transfer of DNA or cells occurs during caprine gestation is critical to effectively protect the public without culling animals that pose no risk. Additionally, fetal-maternal transfer, should it exist in the goat, could contraindicate the rebreeding of nontransgenic dams due to the possible transfer of fetal cells from 1 pregnancy to the fetus of subsequent pregnancies. Fetal-maternal transfer in Capra hircus has not been reported in the literature, although it has been reported in another ruminant, Bos taurus. We examined blood from nontransgenic dams that carried transgenic offspring using a PCR method sensitive enough to detect the presence of a spider silk transgene to a 1:100,000 dilution. At this sensitivity, we did not detect the occurrence of fetal-maternal transfer in 5 nontransgenic dams. Likewise, fetal-fetal transfer was not observed from a transgenic to a nontransgenic twin in utero. To test tissue-specific expression of the silk transgene, proteins purified from standard necropsy tissue from a lactating transgenic dam were examined by Western blot analysis. Silk protein expression was only observed in mammary tissue consistent with the tissue specificity of the ß-casein promoter used in the transgenic construct. We report evidence collected from a limited caprine breeding pool against transfer of transgenes in utero from fetus to dam and fetus to fetus. In addition, we show evidence that the ß-casein promoter in our expression construct is not expressed ectopically as previously suggested. These results suggest that transgene transfer in utero does not occur, but further study is warranted with a larger sample group to confirm these results.

Promoter cloning in the radioresistant bacterium Deinococcus radiodurans.

Deinococcus radiodurans is a highly radiation-resistant bacterium that is classed in a major subbranch of the bacterial domain. Since very little is known about gene expression in this bacterium, an initial study of promoters was undertaken. In order to isolate promoters and study promoter function, a series of integrative vectors for stable chromosomal insertion in D. radiodurans were developed. These vectors are based on Escherichia coli replicons that are unable to replicate autonomously in D. radiodurans and carry homologous sequences for replacement recombination in the D. radiodurans chromosome. The resulting integration vectors were used to study expression of reporter genes fused to a number of putative promoters that were amplified from the D. radiodurans R1 genome. Further analysis of these and other putative promoters was performed by Northern hybridization and primer extension experiments. In contrast to previous reports, the -10 and -35 regions of these promoters resembled the sigma(70) consensus sequence of E. coli.

[Estimation of marker passage in the digestive tract of dairy cows by different models: influence of stage of lactation, amount of intake and diet composition]. / Ermittlung der Passage eines Markers im Verdauungstrakt von Milchkühen mit Hilfe unterschiedlicher Schatzverfahren: Einfluss von Laktationsstadium, Fütterungsniveau und Rationszusammensetzung.

12 dairy cows were used to examine the influence of stage of lactation, amount of intake and diet composition on digesta passage from the reticulorumen. Cows were fed on mixed diets, containing on average 34% concentrate and 66% roughage (dry matter basis). The roughage portion consisted of maize silage and either extensively or intensively produced grass silage in the ratio of 40:60 (% as fed). Titanium(IV)-oxide (TiO2) was used as a marker and faecal marker concentrations were determined for 120 h after oral dosing. Samples were taken in four periods-ante partum (1 period) and in the first half of lactation (3 periods)-, each period representing a certain physiological state. Cows were fed according to milk yield with mean dry matter intakes of 12.0, 16.8, 22.6 and 21.2 kg in periods 1 to 4. Passage parameters were estimated by a model with gamma time-dependency (G4G1), by an exponential model and by a model calculating total tract mean retention time as mean value of all points on the marker-excretion curve. Results showed an obvious acceleration of marker passage from the reticulorumen from ante partum to post partum. Total tract mean retention time calculated as mean values of all points on the marker excretion-curves were in good agreement with total tract mean retention times calculated with G4G1. Passage rates from the reticulorumen showed poor agreement between G4G1 and the exponential model. Effects of period and diet past partum were observed for rate of passage from and mean retention time in the fast compartment only, both estimated by G4G1. TiO2 appears to be a suitable marker to estimate passage rates. Values for passage rates were in the range of those reported for concentrate particles by others.

[Determination of the passage of two markers in the gastrointestinal tract of steers using different evaluation methods: effect of the level of nutrition]. / Ermittlung der passage zweier marker im verdauungstrakt von ochsen mit hilfe unterschiedlicher schätzverfahren: Einfluss des ernährungsniveaus.

This study was conducted to investigate the effects of amount of intake of a mixed diet by steers on the passage of digesta through the reticulorumen and the whole digestive tract. Six ruminally cannulated steers received a mixed diet consisting on average of 43% grass silage, 25% maize silage, 30% concentrate and 2% mineral-vitamin mix in DM. The experimental design was a repeated 3 x 3 Latin square with 21 days periods. The diet was offered twice daily (07.00 and 19.00 h) at 1, 1.5 and 2 times of estimated maintenance energy requirements. At the beginning of each period, animals received pulse doses of Titanium(IV)-oxide (TiO2) per os and Cr-EDTA intraruminally. Following marker administration, faecal marker concentrations were determined over 120 h. Passage parameters were estimated by a mono-exponential and a bi-exponential model, by models including gamma age-dependency and by a model calculating total tract mean retention time as mean value of all points on the marker-excretion curve. Passage rate of TiO2 from the reticulorumen increased with higher intakes, whereas mean retention time in the whole tract decreased. In general, results of different models were consistent across intakes. Values for passage of TiO2 were in good agreement with those reported for small particles, when similar diets were fed to cattle. Rate of passage of Cr-EDTA from the reticulorumen increased with higher intakes, and mean retention times of Cr-EDTA in the reticulorumen and in the whole tract decreased. Differences between models with or without age dependency were greater for Cr-EDTA than for TiO2. Fit to Cr-EDTA excretion curves was not satisfactory for models with gamma age dependency. Irrespective of model and marker, passage from the reticulorumen accelerated markedly, whereas retention times in the reticulorumen and in the whole tract decreased, as intake increased from maintenance energy requirements to 1.5 times maintenance.