Difficulties associated with in vitro manipulation and culture of the early chicken embryo have restricted generation of transgenic chickens to approaches that use replication-competent retroviruses. The need to produce transgenic chickens in the absence of replicating virus prompted development of a new method of gene transfer into the chicken. Microinjection of the replication-defective reticuloendotheliosis virus (REV) vector ME111 beneath unincubated chicken embryo blastoderms results in infection of germline stem cells. This vector contains genetic information exogenous to the chicken genome, including both the herpes simplex virus type 1 thymidine kinase gene and the Tn5 neomycin phosphotransferase gene. About 8 percent of male birds hatched from injected embryos contained vector DNA in their semen. All four positive males tested passed vector sequences onto their progeny. Analysis of G1 offspring showed that gonads of G0 male birds were mosaic with respect to insertion of vector provirus. Thus, primordial germ cells present in the unincubated chicken embryo blastoderm are susceptible to infection by defective REV vectors.
This report describes expression of heritable reticuloendotheliosis virus (REV) vector ME111 in 20 independent lines of transgenic chickens. The results are strikingly different from studies of Moloney virus in transgenic mice, where restricted expression of inherited proviruses has led to their use primarily as insertional mutagens rather than general agents for gene transfer. In contrast, the REV ME111 provirus is actively transcribed in a variety of tissues from transgenic chickens, is expressed from transcriptional control elements present in the long terminal repeat of the provirus, and codes for active neomycin phosphotransferase II. The REV vector system as applied to the chicken represents a departure from the long-established paradigm of retroviral transgenes in mice and provides a new approach to the study of avian biology.Replication-defective vectors derived from reticuloendotheliosis virus (REV) can infect pluripotent stem cells when injected beneath the blastoderm of unincubated chicken eggs (1, 2). Gene transfer at this stage of embryonic development requires the high efficiency of viral infection because the blastoderm contains many thousands of cells (3,4). This procedure gives rise to mosaic chickens which upon subsequent breeding yield transgenic animals hemizygous for unique provirus insertions. Although these viral transgenes are expressed in somatic cells ofthe infected embryo, a major question has been whether inherited provirus would remain transcriptionally active.The first germ-line insertion of experimentally introduced provirus was achieved by infection of the early mouse embryo (5). This and other studies have shown that provirus passed through the mouse germ-line is often transcriptionally inactive (6)(7)(8)(9)(10)(11)(12). The ability to manipulate the mouse embryo has led to alternative methods of gene transfer such as nuclear injection of cloned DNA (13,14) (Fig. la). As a positive control duplicate filters were hybridized with a 1.1-kb Pst I fragment derived from the plasmid ptl, which contains an a-tubulin cDNA homologous with the five-member a-tubulin gene family (24,25). RNA Dot Blot Analysis. Total RNA was denatured in formaldehyde and immobilized on GeneScreenPlus membranes (New England Nuclear). Duplicate RNA samples were treated with 2 M NaOH at 65TC as a control for DNA contamination (data not shown). Filters were baked 60 min at 80TC and prehybridized at 650C for 1 hr in 1 M sodium phosphate, pH 7.2/0.5% SDS/0.1% Ficoll/0.1% polyvinylpyrrolidone/0.1% bovine serum albumin/1 mM EDTA containing 50 gg of denatured salmon sperm DNA per ml (22). A DNA probe (ME111 or a-tubulin; 5 x 106 cpm) was added and filters were hybridized overnight at 650C. Filters were washed twice in 15 mM NaCI/1.5 mM sodium citrate, pH 7/0.2% SDS for 60 min at 65TC and subjected to autoradiography.Northern Blot Analysis. Total RNA was passed once over a Stratagene "quick push" oligo(dT)-cellulose column according to the manufacturer's instructions. Two hundred nanograms of RNA from the ME111 ...
Deficiency in retinoid acid receptor-related orphan receptor alpha (RORα) of staggerer mice results in extensive granule and Purkinje cell loss in the cerebellum as well as in learned motor deficits, cognition impairments and perseverative tendencies that are commonly observed in autistic spectrum disorder (ASD). The effects of RORα on brain lipid metabolism associated with cerebellar atrophy remain unexplored. The aim of this study is to examine the effects of RORα deficiency on brain phospholipid fatty acid concentrations and compositions. Staggerer mice (Rora sg/sg) and wildtype littermates (Rora +/+) were fed n-3 polyunsaturated fatty acids (PUFA) containing diets ad libitum. At 2 months and 7 or more months old, brain total phospholipid fatty acids were quantified by gas chromatography-flame ionization detection. In the cerebellum, all fatty acid concentrations were reduced in 2 months old mice. Since total fatty acid concentrations were significantly different at 2-monthold, we examined changes in fatty acid composition. The composition of ARA was not significantly different between genotypes; though DHA composition remained significantly lowered. Despite cerebellar atrophy at >7months-old, cerebellar fatty acid concentrations had recovered comparably to wildtype control. Therefore, RORα may be necessary for fatty acid accretions during neurodevelopment. Specifically, the effects of RORα on PUFA metabolisms are region-specific and age-dependent.
Objectives Our objective is to examine the effects of lowering dietary omega-6 polyunsaturated fatty acids (n-6 PUFA, linoleic acid; LA) and/or supplementation of omega-3 PUFA (n-3 PUFA, eicosapentaenoic acid and docosahexaenoic acid; EPA and DHA) on voluntary ethanol binge drinking and pathway networks involved in the interactions of PUFA and ethanol in mesolimbic circuit of the brain. Methods Behavioral cohort: Time-pregnant C57BL/6J dams were randomized to one of four custom dietary interventions. The diets varied in the combination of n-6 PUFA (8 energy% LA or 1 en% LA) and n-3 PUFA (0.5 en% EPA + DHA or 0 en% EPA + DHA). The dietary fatty acid compositions are crafted based on contemporary American and evolutionary model intake. Male offspring continued their respective maternal diet for 8 weeks before ethanol exposure. During the 6-week ethanol binge drinking, mice were exposed to cycles of 5-day 20%v/v ethanol for 4 hours in the dark and 2-day abstinence. RNA-Sequencing cohort: Same dietary interventions were applied. 15-week-old male offspring was subjected to a 9-day 2 g/kg and 1-day 5 g/kg ethanol gavage paradigm. Striatum were collected for next generation RNA sequencing after the last dose of gastric gavage. Results Mice fed 1 en% LA and 0 en% EPA + DHA (L6L3) lowered voluntary ethanol binge drinking by 29% as compared to mice fed 8 en% LA and 0 en% EPA + DHA (H6L3) (one-way ANOVA; week 3, p = 0.0072; week 4–6, P < 0.05). Mice fed 1 en% LA and 0.5 en% EPA + DHA (L6H3) or 8 en% LA and 0.5 en% EPA + DHA (H6H3) did not differ in ethanol binge drinking as compared to L6L3 and H6L3 mice. Mice fed L6L3 exhibited more differentially expressed genes in striatum as compared to other dietary interventions (L6L3, 158 genes; H6L3, 16; L6H3, 39; H6H3, 20). Gene set analysis by Panther showed that striatal transcriptomic signatures in response to ethanol gavage are vastly different between dietary interventions. Conclusions Dietary fatty acids may alter striatal transcriptomic response to ethanol which may result in lowering of voluntary ethanol binge drinking. Dietary lowering of n-6 PUFA may be an effective strategy in preventing voluntary alcohol binge drinking which is a major public health concern. Funding Sources National Institutes of Health, National Institute on Alcohol Abuse and Alcoholism, Section of Nutritional Neuroscience.
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