Silencing of fat-1 transgene expression in sheep may result from hypermethylation of its driven cytomegalovirus (CMV) promoter

The fat-1 gene was isolated from roundworm Caenorhabditis elegans, and built into pIRES2-EGFP expression vectors driven by cytomegalovirus (CMV) promoter or cytomegalovirus enhancer and chickenβ-actin (CAG) promoter. Both CMV- and CAG-driven expression vectors were transfected to sheep fetal fibroblast cells. Positive transfected cells were used as donors for somatic cell nuclear transfer (SCNT) and the cloned embryos were transferred into the oviducts of synchronized recipient sheep. Two lambs derived from CMV vector and three lambs derived from CAG vector developed to term. Although Southern analyses using tissues from the two lambs derived from CMV vectors indicated integration of fat-1 gene into the genome, fat-1 mRNAs were not detected by RT-PCR. However, there was fat-1 expression (detected by RT-PCR) in tissues from transgenic lambs driven by CAG vectors. To investigate potential mechanisms involved in the two transgene models, methylation state of the vector promoters were examined. In CMV-driven transgenics, CMV promoters had almost no methylation in transfected cells and the resultant cloned embryos, whereas high methylations were detected in tissues and organs in transgenic lambs. In the CAG-driven transgenics, there were almost no methylations in transgenic cells and transgenic cloned embryos, and cloned lambs expressed fat-1 mRNA (detected by RT-PCR). Moreover, although SV40 promoters which drove neo/kan marker gene in CMV vectors were highly methylated in tissues from transgenic lambs, they were without methylation in cells and embryos. Therefore, we concluded that highly methylated CMV promoters induced the silence of fat-1 transgene expression in sheep. Furthermore, CAG promoter, but not CMV promoter was suitable for generation of fat-1 transgenic sheep.     

Use of the nuclease inhibitor aurintricarboxylic acid (ATA) for improved non-viral intratumoral in vivo gene transfer by jet-injection

BACKGROUND: Stability, integrity and retention of the DNA within the targeted tissue is decisive for efficient gene transfer using naked DNA. Pre-clinical and clinical studies require reproducible transfection rates by preventing rapid degradation of naked DNA in the transduced tissue. Tumor tissues contain nuclease activity, which can affect DNA stability if naked DNA is used. Therefore, inhibition of nuclease-mediated DNA degradation by the nuclease inhibitor aurintricarboxylic acid (ATA) might lead to improved gene transfer efficiency in tumor tissues. METHODS: For both, DNA-degradation analysis and in vivo gene transfer experiments, the beta-galactosidase (LacZ)-expressing pCMVbeta and the cytosine deaminase (CD)-expressing pCMV-CD plasmid were used. Influence of the nuclease inhibitor ATA was determined in tumors, in which naked pCMVbeta or pCMV-CD DNA and ATA was co-administered by jet-injection. The nuclease activity and inhibition by ATA was analyzed using the DNase Alert detection system. The influence of ATA on LacZ expression was determined by specific ELISA and its effect on the therapeutic efficacy of CD gene transfer on tumor growth was determined in vivo. RESULTS: The screening of different human mammary and colon carcinoma models revealed strong nuclease activity rapidly degrading naked plasmid DNA. Co-administration of ATA with pCMVbeta or pCMV-CD for in vivo jet-injection of tumors prevented DNA from nuclease degradation associated with either increased LacZ gene expression or improved reduction in tumor growth. CONCLUSIONS: Tumor-associated nuclease activity is a notable hurdle in gene transfer of naked DNA and therefore inhibition of nucleolytic degradation of plasmid DNA facilitates intratumoral gene expression.     

A review of the effects of the Booroola gene (FecB) on sheep production

Booroola Merino (B^oM) ewes have a high ovulation rate and litter size which in 1980 was postulated to be due to the effects of a major gene (FecB). This was confirmed in breeding experiments and FecB was subsequently shown to be due to a mutation (BMPR-1B) on chromosome 6. The B^oM originated from an Australian commercial fine wool Merino flock (Booroola) and has been used in crossing experiments and for introgression of FecB into many breeds around the world to improve fecundity. The mutation has recently been found in native sheep breeds in India, China and Indonesia and it is likely that FecB in the Australian B^oM was derived from importations of Garole sheep from India in 1792 and 1793.The effects on production traits of the FecB mutation in a range of genetic comparisons, environments and production systems are reviewed. Comparisons involving B^oM crosses with various other breeds and contrasts of FecB homozygous (BB), heterozygous (B+) and non-carrier (++) genotypes in comparable background genotypes, including non-B^oM, have been summarised from 45 reports. The weighted mean effect for ewes carrying one copy of FecB (B+) was +1.3 (range +0.8 to +2.0) for ovulation rate and +0.7 (range +0.4 to +1.3) for litter size. The effect of a second copy (BB) was generally additive for ovulation rate, with little or no increase in litter size for BB ewes among B^oM crosses. However there was generally a further increase in litter size for BB ewes of about half the effect of one copy (B+) in the Indian and Chinese breeds. Poor lamb survival and lamb growth reduced the number of lambs weaned and total weight of lamb weaned by B+ ewes. Most studies still showed a small advantage for B+ ewes, although several reported negative effects. While embryo survival declines at higher ovulation rates, the effects of FecB per se are equivocal. There is some evidence of a higher non-pregnancy rate among homozygous BB ewes. Most studies reported lower birth weight and growth rate from B^oM cross lambs and lambs from crossbred ewes introgressed with FecB. However it is difficult to separate the effects of low background genetic merit for growth of the B^oM and the lower birth weight and growth rate of lambs from larger litters from the genetic effect of carrying FecB. There was little or no difference in growth rate between BB, B+ and ++ genotype lambs. For other traits including, seasonal oestrous activity, carcass and meat quality and wool production, there was no evidence of major effects of FecB. The opportunities for management and nutritional modification of FecB expression and implications for industry adoption are briefly discussed.     

Differential expression of the GTL2 gene within the callipyge region of ovine chromosome 18

The inheritance pattern of the skeletal muscle hypertrophy phenotype caused by the callipyge gene has been characterized as polar overdominance. We hypothesized that this trait may be caused by a gain or loss of gene expression because of the reversible nature of the phenotype in paternal vs. maternal inheritance. Suppression subtraction cDNA probes were made from skeletal muscle mRNA of normal (NN) and callipyge (C(Pat)N(Mat)) animals and hybridized to Southern blots containing bacterial artificial chromosomes (BACs) that comprise a physical contig of the callipyge region. The CN-NN probes hybridized to two ovine and seven bovine BACs. Sequence analysis of fragments within those BACs indicated short regions of similarity to mouse gene trap locus (gtl2). Northern blots analysis of RNA from hypertrophy-responsive muscles show a population of GTL2 mRNA centred around 2.4 kb that were abundantly expressed in 14-day prenatal NN and C(Pat)N(Mat) lambs but were down-regulated in day 14 and day 56 postnatal NN lambs. The expression of GTL2 remained elevated in 14- and 56-day-old C(Pat)N(Mat) lambs as well as in 56-day-old N(Pat)C(Mat) and CC lambs. Expression of GTL2 in the supraspinatus, which does not undergo hypertrophy, was very low for all genotypes and ages. Isolation of cDNA sequences show extensive alternative splicing and a lack of codon bias suggesting that GTL2 does not encode a protein. The mutation of the callipyge allele has altered postnatal expression of GTL2 in muscles that undergo hypertrophy and will help identify mechanisms involved in growth, genomic imprinting and polar overdominance.     

PCR detection of hemolysin (vhh) gene in Vibrio harveyi

The Vibrio harveyi hemolysin gene (vhh), which encodes for a virulence factor involved in pathogenicity to fish and shellfish species, may be targeted for species detection or strain differentiation. Primers designed for this gene were used in detection studies of V. harveyi strains from various hosts. One primer set among four tested, could amplify the expected gene fragment in PCR using templates from all 11 V. harveyi strains studied. Detection of the presence of the hemolysin gene could therefore serve as a suitable detection marker of Vibrio harveyi isolates potentially pathogenic to fish and shrimps.     

Evolution of mercuric reductase (merA) gene: a case of horizontal gene transfer

In the present study the role of horizontal gene transfer events in providing the mercury resistance is depicted. merA is key gene in mer operon and has been used for this study. Phylogenetic analysis of aligned merA sequences shows broad similarities to the established 16S rRNA phylogeny. But there is no separation of bacterial merA from archael merA which suggests that merA gene in both these groups share considerable sequence homology. However, inconsistencies between merA and 16S rRNA gene phylogenetic trees are apparent for some taxa. These discrepancies in the phylogenetic trees for merA gene and 16S rRNA gene have lead to the suggestion that horizontal gene transfer (HGT) is a major contributor for its evolution. The close association among members of different groups in merA gene tree, as supported by high bootstrap values, deviations in GC content and codon usage pattern indicate the possibility that horizontal gene transfer events might have taken place during the evolution of this gene.     

Identifying infertile homozygous Inverdale (FecXI) ewe lambs on the basis of genotype differences in reproductive hormone concentrations

Introduction of the Inverdale prolificacy gene (FecX^I) could markedly improve reproductive efficiency in commercial flocks, but as homozygous carrier Inverdale ewes are infertile, it is imperative that these animals are identified at an early age and excluded from breeding stock. As the ovaries of homozygous carrier ewes are nonfunctional, there are wide differences in reproductive hormone levels between these and other Inverdale genotypes. This study assesses the accuracy of using hormone concentrations alone, to identify infertile homozygous ewe lambs. Ewe lambs were blood sampled at 2, 5 and/or 8 months of age, and plasma analyzed for follicle-stimulating hormone (FSH), luteinizing hormone (LH) and inhibin content. These animals were either the offspring of both known carrier rams and known carrier ewes, and therefore would be either homozygous (II) or heterozygous (I +) for the Inverdale gene (group 1, N = 122), or had one parent that was a carrier and therefore would be either heterozygous or noncarriers (+ +) of the gene (group 2, N = 32). Animals were designated as either II or I + / + + on the basis of their plasma hormone concentrations. Inverdale genotype was also assigned from laparoscopic observation of the ovaries at each of these occasions. Definitive assignment of genotype was made at laparoscopy as adults during the breeding season. On the basis of laparoscopy as adults, 62 (51%) lambs in group 1 were identified as homozygous and 60 (49%) as heterozygous. At all three ages, both mean FSH and mean LH concentrations were significantly higher in II than in I + lambs. Mean inhibin concentrations were significantly lower in II lambs at 8 months, but did not differ significantly between genotypes at 2 or 5 months of age. The use of discriminant analysis techniques to segregate individual animals in group 1 on the basis of their plasma FSH and LH concentrations, correctly identified Inverdale genotype in 50/52 (96%) lambs at 2 months, 75/79 (95%) at 5 months and 118/122 (97%) at 8 months of age. Discriminant analysis was equally effective for segregating II ewe lambs (group 1) from fertile ewe lambs of I + and + + genotype (group 2, 97% correct at 5 months and 98% at 8 months). At no stage did inclusion of inhibin concentrations into the discriminant function alter the number of homozygous ewes misclassified. This demonstrates that infertile homozygous ewe lambs can accurately be distinguished from their fertile flockmates by using plasma concentrations of gonadotrophins alone, and that this can be achieved from as early as 2 months of age.     

Ovarian follicular activity in Booroola lambs with and without a fecundity gene

The ovaries of 3-month-old Booroola lambs which were heterozygous carriers of a major gene (F) influencing the ovulation rate in mature ewes (i.e. F + lambs) were compared to those ofsimilarly-aged Booroola lambs which were non-carriers of the F-gene (i.e. ++ lambs). The ovaries of the F + Booroola lambs were significantly lighter (P less than 0.01) than those of ++ lambs even though the mean +/- s.e.m. number of follicles (greater than or equal to 1 mm diam.) in the F + lambs was greater than that in the ++ lambs (i.e. F + lambs, 30.2 +/- 2.5 follicles; ++ lambs, 18.4 +/- 1.2 follicles; P less than 0.01). In granulosa cells from non-atretic follicles (greater than or equal to 1 mm diam.) from F + and ++ Booroola lambs, FSH (NIAMDD-FSH-S16) doses of 100 and 1000 ng/ml caused significant stepwise increases (P less than 0.05) in cyclic adenosine 3',5'-monophosphate (cAMP) production compared to that achieved at FSH doses of 0 and 1 ng/ml or at any FSH dose in cells from atretic follicles. However, no significant differences in FSH-induced cAMP production were noted with regard to Booroola genotype or follicular diameter. None of the granulosa cell preparations from non-atretic follicles of 1-2.5 mm diameter from F + lambs (N = 13) or from non-atretic follicles of 1-4.5 mm diameter from ++ lambs (N = 16) responded to LH (NIAMDD-LH-S24; 10 or 1000 ng/ml) to produce significantly more cAMP than did the controls. In contrast, the granulosa cell preparations from non-atretic follicles of 3-4.5 mm diameter from F + lambs (N = 4) and from non-atretic follicles of greater than or equal to 5 mm diameter of ++ lambs (N = 4) produced significantly more cAMP (P less than 0.05) in response to LH (1000 and/or 10 ng/ml) relative to that in the controls. The theca interna from follicles of lambs of both genotypes had functional LH receptors as judged by the androstenedione responses to exogenous LH although no genotypic differences were noted. In F + lambs, the follicular fluid concentrations of testosterone but not oestradiol (i.e. in 1-4.5 mm diam. follicles) and granulosa cell aromatase activity (i.e. in 3-3.5 mm diam. follicles) were significantly higher (both P less than 0.05) than in corresponding follicles or cells from ++ lambs. Collectively the results suggest that the Booroola F-gene influences the composition and function of sheep ovaries before puberty.     

Rate of abnormalities in lambs from in vitro produced embryos transferred on Day 2 compared with Day 6 postfertilization

The effect of transferring ovine IVP embryos on Day 2 versus Day 6 postinsemination was investigated. Oocytes were collected from 35 cull ewes and cultured separately for each donor. Embryos were exposed to serum in the maturation and fertilization media, and then cultured in a serum-free SOF system under serum-conditioned silicone oil. Cleaved Day 2 postfertilization embryos were randomly allocated for immediate transfer versus Day 6 transfer. Parturition was induced (with 15 mg dexamethasone) between Days 141 and 143 of pregnancy; those failing to initiate parturition by Day 146 had a Caesarean section. Stillborn lambs and those dying within several days of birth were necropsied. Lambs were weighed at birth; lambs (n = 132) derived from the same flock (from natural breeding or AI) were used as a control. An estimate of the 99th percentile of the normal population was used as the point above which lambs from three litter-size categories were classified as abnormally large (analysis was repeated for 95% confidence and median estimates of this value). Thirty-eight lambs were born from early transfer, 45 from late transfer and 12 from unobserved lambings (parentage could not be determined). Lambs from early transfer were heavier at birth (P = 6.0 x 10 (-11)). The early treatment group had fewer lambs produced per embryo transferred (P = 0.0002), fewer live lambs per embryo transferred (P = 0.00009) and fewer normal lambs per embryo transferred (P= 0.0002). There was an effect of donor on the number of dead lambs per embryo transferred (P = 0.017). The number of dystocias per transfer was not significantly different for early versus late transfer groups. However, the probabilities of survival for cleaved embryos were 27.3% and 13.1% in the early and late transfer treatment, respectively. There were more lambs produced per cleaved embryo with early transfer (P = 0.004) and there was an effect of donor within ram and replicate (P = 0.04) on the number of lambs produced. The odds ratio for early/late treatment was 2.449 with a confidence interval of 1.368-4.382. Day 2 transfer of cleaved embryos did not prevent the production of oversized or abnormal offspring; however, there was an increased chance of a cleaved embryo producing a lamb when transferred on Day 2 rather than Day 6 postfertilization.     

Targeted vectors for gene therapy of cancer and retroviral infections

Gene therapy has developed to a technology which rapidly moved from the laboratory bench to the bedside in the clinic. This implies safe, efficient and targeted gene transfer systems for suitable application to the patient. Beside the development of such gene transfer vectors of viral or nonviral origin, improvement of cell type specific and inducible gene expression is pivotal for successful gene therapy leading to targeted gene action. Numerous gene therapy approaches for treatment of cancer and retroviral infections utilize cell type specific and/or regulatable promoter and enhancer sequences for the selective expression of therapeutic genes in the desired cell populations and tissues. In this article the recent developments and the potential of expression targeting are reviewed for gene therapy approaches of cancer and retroviral infections.     

Possible mechanism of polycation liposome (PCL)-mediated gene transfer

A novel gene transfer system utilizing polycation liposomes (PCLs), obtained by modifying liposomes with cetyl polyethylenimine (PEI), was previously developed (Gene Ther. 7 (2002) 1148). PCLs show notable transfection efficiency with low cytotoxicity. However, the mechanism of PCL-mediated gene transfer is still unclear. In this study, we examined the intracellular trafficking of PCL-DNA complexes by using HT1080 cells, fluorescent probe-labeled materials, and confocal laser scan microscopy. We found that the PCL-DNA complexes were taken up into cells by the endosomal pathway, since both cellular uptake of the complex and gene expression were blocked by wortmannin, an inhibitor of this pathway. We also observed that the plasmid DNA and cetyl PEI complex became detached from the PCL lipids and was preferentially transferred into the nucleus in the form of the complex, whereas the PCL lipids remained in the cytoplasmic area, possibly in the endosomes. In fact, nigericin, which dissipates the pH gradient across the endosomal membrane, inhibited the detachment of lipids from the PCL-DNA complex and subsequent gene expression. Taken together, our data indicate the following mechanism for gene transfer by PCLs: PCLs effectively transfer DNA to endosomes and release cetyl PEI-DNA complexes into the cytosol. Furthermore, cetyl PEI also contributes to gene entry into the nucleus.     

Iodine supplementation of the pregnant dam alters intestinal gene expression and immunoglobulin uptake in the newborn lamb

Excess iodine intake by the pregnant dam reduces lamb serum antibody concentration, specifically immunoglobulin G (IgG). An experiment was conducted to investigate the mechanisms under pinning the reduced serum IgG concentration at 24 h postpartum in the progeny of iodine supplemented dams. Forty-five mature twin bearing ewes (n=15/treatment) were allocated to one of three dietary treatments as follows: basal diet (Control); basal diet plus 26.6 mg of iodine per ewe per day as calcium iodate (CaIO₃); or potassium iodide (KI). Ewes were individually housed and fed from d 119 of gestation until parturition. All lambs received colostrum at 1, 10 and 18 h postpartum via stomach tube. At 1 h postpartum lambs from the control and an iodine supplemented treatment (n=10 per treatment from control and CaIO₃) were euthanised before colostrum consumption and ileal segments isolated to determine the gene expression profile of a panel of genes identified as having a role in antibody transfer. Preceding euthanasia, lambs were blood sampled for determination of serum IgG, total thyroxine and free tri-iodothyronine concentrations. Progeny of CaIO₃ supplemented dams had lower tri-iodothyronine concentrations (P<0.01) at 1 h postpartum and lower serum IgG concentrations (P<0.001) at 24 h postpartum when compared with the progeny of control dams. Iodine (CaIO₃) supplementation of the dam increased the relative expression (P<0.05) of the B2M, PIGR and MYC genes in the ileum of the lamb, before colostrum consumption; while the expression of THRB declined when compared with the progeny of C dams (P<0.01). In conclusion, the results of this study show that it is the actual inclusion of excess iodine in the diet of the ewe, regardless of the carrier element, that negatively affects passive transfer in the newborn lamb. This study presents novel data describing the relationship between maternal iodine nutrition and its effect on the thyroid hormone status and subsequent gene expression in the newborn lamb; which results in a failure of passive transfer and a decline in serum IgG concentration.     

Experimental Infection of Lambs with an Equine Granulocytic Ehrlichia Species Resembling the Agent that Causes Human Granulocytic Ehrlichiosis (HGE)

Five lambs were inoculated with a granulocytic Ehrlichia species originally isolated from a Swedish horse with granulocytic ehrlichiosis (EGE). The 16S rRNA gene sequence of the Swedish Ehrlichia sp. causing EGE was identical to the sequence of the agent causing human granulocytic ehrlichiosis (HGE). After the inoculation, infected neutrophils and a low serologic response were seen in all lambs, but no clinical symptoms were observed. In one lamb 17% of the neutrophils were infected without a corresponding fever. Six weeks later the lambs were inoculated with an ovine isolate of E. phagocytophila. After challenge with E. phagocytophila the lambs reacted with fever and infected granulocytes. The results presented herein show that the equine Ehrlichia isolate was infective for lambs but generated weak immune response and no distinctive protection from subsequent challenge with E. phagocytophila.     

Percutaneous in vivo gene transfer to the peripheral lungs using plasmid-liposome complexes

The purpose of this study was to investigate a new method of in vivo gene transfer to the lung parenchyma by the percutaneous approach. The plasmid that contains the gene for firefly luciferase driven by a cytomegalovirus (CMV) promoter (pCMVL) in combination with cationic lipids was percutaneously injected into the lung parenchyma. Luciferase activities were localized to the lobes of the lung where the plasmids with cationic lipids were injected. Percutaneous injection of the plasmid containing the human endothelin-1 (hET-1) gene driven by a CMV promoter (pRc/CMVhET-1) in combination with cationic lipids into the lungs caused pulmonary fibrosis localized to the injection site in the peripheral lungs. We concluded that percutaneous in vivo gene transfer to the lungs is a unique and important approach to introduce exogenous gene expression in the limited area of the lung parenchyma. This method of gene transfer will be applicable for human gene therapy for targeted areas of peripheral lung and will also be useful to assess the function of the proteins expressed by a gene in the local area of the lungs.     

Fibrolytic Bacteria Isolated from the Rumen of North American Moose (Alces alces) and Their Use as a Probiotic in Neonatal Lambs

Fibrolytic bacteria were isolated from the rumen of North American moose (Alces alces), which eat a high-fiber diet of woody browse. It was hypothesized that fibrolytic bacteria isolated from the moose rumen could be used as probiotics to improve fiber degradation and animal production. Thirty-one isolates (Bacillus, n = 26; Paenibacillus, n = 1; and Staphylococcus, n = 4) were cultured from moose rumen digesta samples collected in Vermont. Using Sanger sequencing of the 16S rRNA gene, culturing techniques, and optical densities, isolates were identified and screened for biochemical properties important to plant carbohydrate degradation. Five isolates were selected as candidates for use as a probiotic, which was administered daily to neonate lambs for 9 weeks. It was hypothesized that regular administration of a probiotic to improve fibrolysis to neonate animals through weaning would increase the developing rumen bacterial diversity, increase animal production, and allow for long-term colonization of the probiotic species. Neither weight gain nor wool quality was improved in lambs given a probiotic, however, dietary efficiency was increased as evidenced by the reduced feed intake (and rearing costs) without a loss to weight gain. Experimental lambs had a lower acetate to propionate ratio than control lambs, which was previously shown to indicate increased dietary efficiency. Fibrolytic bacteria made up the majority of sequences, mainly Prevotella, Butyrivibrio, and Ruminococcus. While protozoal densities increased over time and were stable, methanogen densities varied greatly in the first six months of life for lambs. This is likely due to the changing diet and bacterial populations in the developing rumen.     

Effects of the FecL major gene in the Lacaune meat sheep population

Background

The major prolificacy gene FecL was first described in the Lacaune sheep meat breed Ovi-Test in 1998. A few studies estimated the effect of this gene on prolificacy but little data is available. In 2010, the Ovi-Test cooperative started genotyping FecL in all of their replacement ewe lambs. Thanks to the large amount of genotyping data that is available now, gene effects on litter size and other relevant traits can be estimated more accurately.

Methods

Our study included 5775 ewes genotyped since 2010 and 1025 sires genotyped since 2002. Performances and pedigrees were extracted from the French national database for genetic evaluation and research. Analysis of the effect of the gene on different traits was based on linear or threshold genetic animal models using the ASReml software.

Results

The female population was composed of 71% homozygous wild type ewes (++), 27% heterozygous ewes for the FecL mutation (L+) and 2% homozygous mutant (LL) ewes. On average, L + ewes produced 0.5 more lambs per lambing than ++ ewes. The FecL gene not only affected the mean litter size but also its variability, which was lower for ++ than for L + ewes. Fertility after insemination was higher for L + ewes than for ++ ewes. Lambs from ++ dams were heavier (+300 g) than the lambs of L + dams and the mortality of twin lambs born from ++ dams was lower than those from L + dams. In addition, bias in estimated breeding values for prolificacy when ignoring the existence of this major gene was quantified.

Conclusions

The effect of the FecL gene on prolificacy was estimated more accurately and we show that this gene affects both the mean and the variability of litter size and other traits. This paper also shows that ignoring the existence of this major gene in genetic evaluation of prolificacy can lead to a large overestimation of polygenic breeding values.     

A mutation in gene CNGA3 is associated with day blindness in sheep

Lambs with congenital day blindness show diminished cone function, which is characteristic of achromatopsia, a congenital disorder described in humans and dogs. To identify gene(s) associated with sheep day blindness, we investigated mutations in the CNGA3, CNGB3, and GNAT2 genes which have been associated with achromatopsia. Sequencing the coding regions of those genes from four affected and eight non-affected lambs showed that all affected lambs were homozygous for a mutation in the CNGA3 gene that changes amino acid R236 to a stop codon. By PCR-RFLP-based testing, homozygosity for the stop codon mutation was detected in another 19 affected lambs. Non-affected individuals (n = 386) were non-carriers or heterozygous for the mutation. While a selection program has been launched to eradicate the day blindness mutation from Improved Awassi flocks, a breeding nucleus of day-blind sheep has been established to serve as animal models for studying human achromatopsia.     

Establishment of a rapid and scalable gene expression system in livestock by site-specific integration

Somatic cell-mediated transgenesis is routinely used to transfer exogenous genes to livestock genomes. However, transgene insertion events are essentially random which may lead to transgene silencing or alter animal phenotype because of insertional mutagenesis. To overcome these problems, we established a gene manipulation system in goat somatic cells based on homologous recombination and flp recombinase-mediated site-specific integration. First, we performed gene targeting to introduce an frt-docking site into the α1 (I) procollagen (ColA1) locus in goat somatic cells. Second, the targeted cell clones were rejuvenated by embryo cloning, and the vigorous cells with targeted frt were reestablished. Third, a gene-replacement system was used to introduce an EGFP reporter gene into the targeted ColA1 locus via flp mediated recombination. As a result, the transgenic somatic cell exhibited faithful expression of EGFP gene under control of the CMV promoter. Similarly, other expression vectors can be introduced into the defined site to evaluate gene functions or express valuable proteins. The gene manipulation system described here will be applicable in other livestock somatic cells, and would allow for the rapid generation of livestock with transgene targeted to the defined site.     

Species-wide variation in the Escherichia coli flagellin (H-antigen) gene

Escherichia coli is a clonal species. The best-understood components of its clonal variation are the flagellar (H) and polysaccharide (O) antigens, both well documented since the mid-1930s because of their use in serotyping. Flagellin is the protein subunit of the flagellum that carries H-antigen specificity. We show that 43 of the 54 H-antigen specificities of E. coli map to the flagellin gene at fliC and sequenced all 43 forms and confirmed specificity of each by cloning and expression. This is, to our knowledge, the first time that all known forms of such a highly polymorphic gene have been fully sequenced and characterized for any species. The established distinction between a highly variable central region and more conserved flanking regions is upheld. The sequences fall into two groups, one of which may be derived from the fliC gene of the E. coli/Salmonella enterica common ancestor, the other perhaps obtained by lateral transfer since species divergence. Comparison of sequences revealed that both horizontal DNA transfer and fixation of mutations under diversifying selection pressure contributed to polymorphism in this locus.     

A high throughput targeted gene disruption method for Alternaria brassicicola functional genomics using linear minimal element (LME) constructs

Alternaria brassicicola causes black spot disease of cultivated Brassicas and has been used consistently as a necrotrophic fungal pathogen for studies with Arabidopsis. In A. brassicicola, mutant generation has been the most rate-limiting step for the functional analysis of individual genes due to low efficiency of both transformation and targeted integration. To improve the targeted gene disruption efficiency as well as to expedite gene disruption construct production, we used a short linear construct with minimal elements, an antibiotic resistance selectable marker gene, and a 250- to 600-bp-long partial target gene. The linear minimal element (LME) constructs consistently produced stable transformants for diverse categories of genes. Typically, 100% of the transformants were targeted gene disruption mutants when using the LME constructs, compared with inconsistent transformation and usually less than 10% targeted gene disruption with circular plasmid disruption constructs. Each mutant displayed a unique molecular signature thought to originate from endogenous exonuclease activities in fungal cells. Our data suggests that a DNA double-stranded break repair mechanism (DSBR) functions to increase targeting efficiency. This method is advantageous for high throughput gene disruption, overexpression, and reporter gene introduction within target genes, especially for asexual filamentous fungi where genetic approaches are unfavorable.