Transgene integration patterns and expression levels in transgenic tissue lines of Picea mariana,P. glauca and P. abies

Seventy transgenic tissue lines (translines) of three spruce species ( Picea mariana, P. glauca and P. abies) were characterized with respect to the integration pattern of the gus (beta-glucuronidase) gene, and the level of GUS activity was determined in 81 lines. The majority of the P. mariana translines (18 lines of 22) integrated multicopies of the transgene, whereas mostly single integrations were detected in the other two species. The activity levels of GUS varied widely among the individual translines of P. mariana, and there was a strong indication that the logarithm of GUS activity increased with the number of gus copies ( P=0.0003) in lines with one to five known insertions (uncensored). The average level of GUS activity, in lines that integrated one gene copy, was the highest in white spruce followed by black spruce and Norway spruce (22.7, 16.5 and 6.3 nmol 4-methylumbelliferone min(-1 )mg(-1 )protein, respectively).     

Normal calves from transfer of biopsied, sexed and vitrified IVP bovine embryos

Data on biopsied, sexed and cryopreserved in vitro produced (IVP) bovine embryos, and their in vivo developmental competence are very limited. Two preliminary studies were conducted before the primary study. In Experiment 1, post-thaw in vitro developmental competence of biopsied and vitrified IVP embryos was evaluated using re-expansion as an endpoint. In Experiment 2, the pregnancy rates of biopsied fresh, frozen or vitrified embryos following single embryo transfer were compared. Since vitrified embryos resulted in a higher pregnancy rate than frozen-thawed embryos, in the primary study (Experiment 3), all IVP embryos were vitrified following biopsy and sexing (by DNA fingerprinting). In Experiment 3, we compared pregnancy initiation and calving results of heifers in the following treatments: 1) artificial insemination (AI); 2) AI plus contralateral transfer of a single embryo (AI + SET); 3) ipsilateral transfer of single embryo (SET); or 4) bilateral transfer of two embryos (DET). Birth weights, gestation lengths and dystocia scores were recorded. In Experiment 1, post-thaw re-expansion rate of biopsied and vitrified embryos was 85% (70/82). In Experiment 2, pregnancy rates (90 d) were 44% (7/16), 23% (3/13), and 50% (7/14) for vitrified, frozen and fresh embryos, respectively (P < 0.10). In Experiment 3, pregnancy rates of AI and SET were 65% (20/31) and 40% (16/40), respectively (P < 0.05). The pregnancy rate of AI + SET was 75% (27/36) with 11 carrying twins, and the pregnancy rate of DET was 72% (26/36) with 10 carrying twins. All AI fetuses were carried to term, but only half the SET fetuses were carried to term. Similar calving rates were observed in the AI + SET and DET groups, 76 and 70%, respectively, of those pregnant at Day 40. Mean birth weight, dystocia score and gestation length of AI calves were not different from those of SET calves. Mean birth weight and dystocia score of single-born calves were greater than those of twin born calves (P < 0.05). These data demonstrate that biopsied IVP bovine embryos can be successfully cryopreserved by vitrification and following post-thaw embryo transfer, acceptable rates of offspring with normal birth weights can be obtained without major calving difficulties.     

Dynamic rearrangement of the filamentous actin network occurs during zoosporogenesis and encystment in the oomycete phytophthora cinnamomi

The organization of filamentous actin (F-actin) in living cells of the oomycete Phytophthora cinnamomi was determined during zoosporogenesis and zoospore encystment by microinjecting sporangia with fluorescently labeled phalloidin and observing resultant fluorescence by confocal microscopy. In multinucleate sporangia prior to the induction of cleavage, phalloidin labeling took the form of plaques which occurred mainly in the periphery of the sporangia. After induction of cleavage, phalloidin labeling showed that the plaques disappeared and that F-actin began to accumulate along the developing cleavage planes and around nuclei and water expulsion vacuoles. F-actin labeling was also observed near the plasma membrane in zoospores and young cysts but reverted to the plaque form in older cysts. Localization of F-actin close to the developing cleavage planes is consistent with the idea that actin microfilaments function in the positioning and expansion of the cleavage membranes. Observations of plaques of actin in living sporangia provide evidence that plaques are not aldehyde-induced fixation artifacts. Copyright 1998 Academic Press.     

Serglycin expression during monocytic differentiation of U937-1 cells

Serglycin is the major proteoglycan in most hematopoietic cells, including monocytes and macrophages. The monoblastic cell line U937-1 was used to study the expression of serglycin during proliferation and differentiation. In unstimulated proliferating U937-1 cells serglycin mRNA is nonconstitutively expressed. The level of serglycin mRNA was found to correlate with the synthesis of chondroitin sulfate proteoglycan (CSPG). The U937-1 cells were induced to differentiate into different types of macrophage-like cells by exposing the cells to PMA, RA, or VitD3. These inducers of differentiation affected the expression of serglycin mRNA in three different ways. The initial upregulation seen in the normally proliferating cells was not observed in PMA treated cells. In contrast, RA increased the initial upregulation, giving a reproducible six times increase in serglycin mRNA level from 4 to 24 h of incubation, compared to a four times increase in the control cells. VitD3 had no effect on the expression of serglycin mRNA. The incorporation of (35S)sulfate into CSPG decreased approximately 50% in all three differentiated cell types. Further, the (35S)CSPGs expressed were of larger size in PMA treated cells than controls, but smaller after RA treatment. This was due to the expression of CSPGs, with CS-chains of 25 and 5 kDa in PMA and RA treated cells, respectively, compared to 11 kDa in the controls. VitD3 had no significant effect on the size of CSPG produced. PMA treated cells secreted 75% of the (35S)PGs expressed, but the major portion was retained in cells treated with VitD3 or RA. The differences seen in serglycin mRNA levels, the macromolecular properties of serglycin and in the PG secretion patterns, suggest that serglycin may have different functions in different types of macrophages.      

Response to selection for litter size in Danish Landrace pigs: a Bayesian analysis

A replicated selection experiment aimed at increasing litter size (total number of pigs born per litter) in Danish Landrace pigs was conducted from 1984 to 1991. The experiment included two selection and two control lines. In each generation, 30 and 14 first litters were produced in selection and control lines, respectively, and dams produced two litters. Each replicate, consisting of one selection and one control line, was founded from 60 families chosen randomly from the population at large. Family selection was practiced, and the criterion was the predicted breeding value for litter size computed using a repeatability animal model, and taking into account all available information. The data consisted of 947 records from 523 dams (424 dams had two litters) representing five cycles of selection of increased litter size. Data were analyzed from a Bayesian perspective, based on marginal posterior distributions of genetic parameters of interest. Marginalization was achieved using Gibbs sampling, with a single chain length of 1 205 000. After discarding the first 5 000 iterations, a sample was drawn every ten iterations, so 120 000 samples in total were saved. Densities were estimated and plotted, and summary statistics were computed from the estimated densities. The posterior means (± standard error) of heritability and repeatability were 0.22 ± 0.06 and 0.32 ± 0.05, respectively. These point estimates of genetic parameters were within the range of literature values, although on the high side. The posterior mean (± standard error) of genetic response to selection, defined as the difference between the mean breeding values of the selected lines and that of the base population, was 1.37 ± 0.43 pigs after five cycles of selection. The regression (through the origin) of breeding values in the selected lines on generation was 0.25 ± 0.08 pigs. Several informative priors constructed from information obtained with field data in this population were used to examine their influence on inferences. The priors were influential because of the relatively small scale of the experiment. An analysis excluding data from one of the control lines gave smaller genetic variance and heritability, and a smaller response to selection. However, it appears that selection for litter size is effective, but that the true rate of response is probably smaller than data from this experiment suggest.     

Effects of Ancestral X Irradiation Followed by Random Mating on Body Weight of Rats

Effects of nine generations of 450r per generation of ancestral spermatogonial X irradiation of inbred rats on body weight were examined. After six generations of random mating (avoiding inbreeding) following the termination of irradiation, descendants of irradiated males (R) were significantly lighter than their controls (C) at 3 and 6 weeks, but not at 10 weeks of age. However, differences in growth between R and C populations were small. Among-litter and within-litter variance estimates were generally larger in the R lines than in the C lines, suggesting that selection responses would be greater in R than in C lines. In conjunction with previous evidence—obtained during the irradiation phase of the experiment—this suggested that more rapid response to selection for 6-week body weight, in particular, might accrue in the R lines.     

Repeat transduction in the mouse lung by using adeno-associated virus vectors with different serotypes

Vectors derived from adeno-associated virus type 2 (AAV2) promote gene transfer and expression in the lung; however, we have found that while gene expression can persist for at least 8 months in mice, it was reduced dramatically in rabbits over a period of 2 months. The efficiency and persistence of AAV2-mediated gene expression in the human lung have yet to be determined, but it seems likely that readministration will be necessary over the lifetime of an individual. Unfortunately, we have found that transduction by a second administration of an AAV2 vector is blocked, presumably due to neutralizing antibodies generated in response to the primary vector exposure. Here, we have explored the use of AAV2 vectors pseudotyped with capsid proteins from AAV serotypes 2, 3, and 6 for readministration in the mouse lung. We found that an AAV6 vector transduced airway epithelial and alveolar cells in the lung at rates that were at least as high as those of AAV2 pseudotype vectors, while transduction rates mediated by AAV3 were much lower. AAV6 pseudotype vector transduction was unaffected by prior administration of an AAV2 or AAV3 vector, and transduction by an AAV2 pseudotype vector was unaffected by prior AAV6 vector administration, showing that cross-reactive neutralizing antibodies against AAV2 and AAV6 are not generated in mice. Interestingly, while prior administration of an AAV2 vector completely blocked transduction by a second AAV2 pseudotype vector, prior administration of an AAV6 vector only partially inhibited transduction by a second administration of an AAV6 pseudotype vector. Analysis of sera obtained from mice and humans showed that AAV6 is less immunogenic than AAV2, which helps explain this finding. These results support the development of AAV6 vectors for lung gene therapy both alone and in combination with AAV2 vectors.