Bacterial Artificial Chromosome Research Articles

Cardiometabolic Consequences of Deleting the Regulator of G protein Signaling-2 (Rgs2) From Cells Expressing Agouti-Related Peptide or the ANG (Angiotensin) II Type 1A Receptor in Mice.

RGS (regulator of G protein signaling) family members catalyze the termination of G protein signaling cascades. Single nucleotide polymorphisms in the RGS2 gene in humans have been linked to hypertension, preeclampsia, and anxiety disorders. Mice deficient for Rgs2 (Rgs2Null) exhibit hypertension, anxiety, and altered adipose development and function. To study cell-specific functions of RGS2, a novel gene-targeted mouse harboring a conditional allele for the Rgs2 gene (Rgs2Flox) was developed. These mice were bred with mice expressing Cre-recombinase via the Agouti-related peptide locus (Agrp-Cre) to cause deletion of Rgs2 from all cells expressing Agrp (Rgs2Agrp-KO), or a novel transgenic mouse expressing Cre-recombinase via the ANG (angiotensin) type 1A receptor (Agtr1a/ AT1A) promoter encoded in a bacterial artificial chromosome (BAC-AT1A-Cre) to delete Rgs2 in all Agtr1a-expressing cells (Rgs2AT1A-KO). Whereas Rgs2Flox, Rgs2Agrp-KO, and BAC-AT1A-Cre mice exhibited normal growth and survival, Rgs2AT1A-KO exhibited pre-weaning lethality. Relative to littermates, Rgs2Agrp-KO exhibited reduced fat gains when maintained on a high fat diet, associated with increased energy expenditure. Similarly, surviving adult Rgs2AT1A-KO mice also exhibited increased energy expenditure. Surprisingly, given the hypertensive phenotype previously reported for Rgs2Null mice and evidence supporting a role for RGS2 in terminating AT1A signaling in various cell types, Rgs2AT1A-KO mice exhibited normal blood pressure, ingestive behaviors, and renal functions, both before and after chronic infusion of ANG (490 ng/kg/min, sc). These results demonstrate the development of a novel mouse with conditional expression of Rgs2 and illustrate the role of Rgs2 within selected cell types for cardiometabolic control.

A transgenic bacterial artificial chromosome approach to identify regulatory regions that direct Amhr2 and Osterix expression in Müllerian duct mesenchyme.

A transgenic mouse approach using bacterial artificial chromosomes (BAC) was used to identify regulatory regions that direct Müllerian duct expression for Amhr2 and Osterix (Osx, also known as Sp7). Amhr2 encodes the receptor that mediates anti-Müllerian hormone (AMH) signaling for Müllerian duct regression in male embryos. Amhr2 is expressed in the Müllerian duct mesenchyme of both male and female embryos. A ∼147-kb BAC clone containing the Amhr2 locus was used to generate transgenic mice. The transgene was able to rescue the block in Müllerian duct regression of Amhr2-null males, suggesting that the BAC clone contains regulatory sequences active in male embryos. Osx is expressed in the developing skeleton of male and female embryos but is also an AMH-induced gene that is expressed in the Müllerian duct mesenchyme exclusively in male embryos. Osx-Cre transgenic mice were previously generated using a ∼204-kb BAC clone. Crosses of Osx-Cre mice to Cre-dependent lacZ reporter mice resulted in reporter expression in the developing skeleton and in the Müllerian duct mesenchyme of male but not female embryos. Osx-Cherry transgenic mice were previously generated using a 39-kb genomic region surrounding the Osx locus. Osx-Cherry embryos expressed red fluorescence in the developing skeleton and Müllerian duct mesenchyme of male but not female embryos. In addition, female Osx-Cherry embryos ectopically expressing human AMH from an Mt1-AMH transgene activated red fluorescence in the Müllerian duct mesenchyme. These results suggest that the 39-kb region used to generate Osx-Cherry contains male-specific Müllerian duct mesenchyme regulatory sequences that are responsive to AMH signaling. These BAC transgenic mouse approaches identify two distinct regions that direct Müllerian duct mesenchyme expression and contribute fundamental knowledge to define a gene regulatory network for sex differentiation.

Sex chromosome differentiation via changes in the Y chromosome repeat landscape in African annual killifishes Nothobranchius furzeri and N. kadleci.

Homomorphic sex chromosomes and their turnover are common in teleosts. We investigated the evolution of nascent sex chromosomes in several populations of two sister species of African annual killifishes, Nothobranchius furzeri and N. kadleci, focusing on their under-studied repetitive landscape. We combined bioinformatic analyses of the repeatome with molecular cytogenetic techniques, including comparative genomic hybridization, fluorescence in situ hybridization with satellite sequences, ribosomal RNA genes (rDNA) and bacterial artificial chromosomes (BACs), and immunostaining of SYCP3 and MLH1 proteins to mark lateral elements of synaptonemal complexes and recombination sites, respectively. Both species share the same heteromorphic XY sex chromosome system, which thus evolved prior to their divergence. This was corroborated by sequence analysis of a putative master sex determining (MSD) gene gdf6Y in both species. Based on their divergence, differentiation of the XY sex chromosome pair started approximately 2 million years ago. In all populations, the gdf6Y gene mapped within a region rich in satellite DNA on the Y chromosome long arms. Despite their heteromorphism, X and Y chromosomes mostly pair regularly in meiosis, implying synaptic adjustment. In N. kadleci, Y-linked paracentric inversions like those previously reported in N. furzeri were detected. An inversion involving the MSD gene may suppress occasional recombination in the region, which we otherwise evidenced in the N. furzeri population MZCS-121 of the Limpopo clade lacking this inversion. Y chromosome centromeric repeats were reduced compared with the X chromosome and autosomes, which points to a role of relaxed meiotic drive in shaping the Y chromosome repeat landscape. We speculate that the recombination rate between sex chromosomes was reduced due to heterochiasmy. The observed differences between the repeat accumulations on the X and Y chromosomes probably result from high repeat turnover and may not relate closely to the divergence inferred from earlier SNP analyses.

Generation of a DNA-launched classical swine fever virus infectious clone packaged in bacterial artificial chromosome

Reverse genetics system offers powerful tool for the research of RNA viruses. The infectious clones of classical swine fever virus (CSFV) were commonly constructed either in high- or low-copy number plasmids and transcribed to infectious RNA using phage RNA-polymerases. Herein, the full-length genome of CSFV Shimen strain, flanked by cytomegalovirus immediate-early (CMV) promoter (a eukaryotic RNA polymerase II promoter) sequence at the 5′-end and the hepatitis delta virus ribozyme along with the bovine growth hormone termination and polyadenylation signal sequences at the 3′-end, was packaged in bacterial artificial chromosome vector to establish a CSFV infectious clone pBAC-smCSFV. This infectious cDNA clone maintained stability after passaged 20 times in bacteria. Transfection of PK15 cells with this cDNA clone facilitated recovery of infectious progeny virus which was identical to parent virus as characterized by RT-qPCR, western blotting, indirect immunofluorescence assay, one-step growth kinetics analysis and nucleotide sequencing. Based on this CSFV infectious cDNA clone, the mCherry was inserted between viral Npro and C protein to develop reporter virus CSFV-mCherry. The mCherry was stably expressed after CSFV-mCherry was passaged 10 times in PK15 cells. Taken together, this present study develops a concise and efficient CSFV infectious cDNA clone and a reporter virus CSFV-mCherry. To the best of our knowledge, this is the first combination of CMV promoter and BAC system in construction of CSFV reverse genetics system. The CSFV infectious cDNA clone and the reporter virus will be useful in the study of CSFV virus biology, virulence determinants, molecular pathogenesis, vaccine development and virus-host interaction.

Epigenetic master regulators HDAC1 and HDAC5 control pathobiont Enterobacteria colonization in ileal mucosa of Crohn’s disease patients

ABSTRACT Adherent-invasive Escherichia coli (AIEC), which abnormally colonize the ileal mucosa of Crohn’s disease (CD) patients, are known to contribute to the etiopathogenesis of CD. Molecular mechanisms favoring AIEC ileal colonization have not been completely characterized yet. The aim of this study was to investigate whether epigenetic regulators histone deacetylases (HDAC) expression in intestinal epithelial cells of CD patients regulate Enterobacteria and AIEC encroachment to intestinal mucosa. HDAC were inhibited in vitro and in CEABAC10 mice to decipher their involvement in the entry of AIEC within host cells. CD ileal samples from the REMIND cohort were used to study the relationship between HDAC expression level and Enterobacteria/AIEC colonization in patients. Mice were fed a westernized diet and orally challenged with AIEC to determine the impact of diet on HDAC expression. Global level of acetylated histone H3 is higher in patients colonized by AIEC bacteria compared to patients non-colonized by Enterobacteria and HDAC inhibition-mediated H3 hyperacetylation promotes the entry of AIEC bacteria within intestinal epithelial cells. HDAC1 and HDAC5 are central and antagonistic in the regulation of AIEC entry within host cells in vitro, in mouse models and in ileal mucosa of CD patients. In mice fed a western-type diet, AIEC infection decreases HDAC1 expression, inducing H3 hyperacetylation to favor their own colonization. CD patients under a western diet are more prone to be colonized by AIEC bacteria as such a diet affects intestinal homeostasis, enables AIEC access to intestinal mucosa where they then manipulate host-epigenome to their advantage. Abbreviations AIEC Adherent-Invasive Escherichia coli; BSA Bovine serum albumin; CD Crohn’s disease; CEABAC10 Carcinoembryonic antigen bacterial artificial chromosome 10; CEACAM Carcinoembryonic antigen-related cell adhesion molecule; FBS Fetal bovine serum; IBD Inflammatory Bowel Disease; HAT Histone acetyltransferase; HDAC Histone deacetylase; kDa KiloDalton; SAHA Suberoylanilide Hydroxamic Acid; Scr Scramble

Arms race and fluctuating selection dynamics in Pseudomonas aeruginosa bacteria coevolving with phage OMKO1.

Experimental evolution studies have examined coevolutionary dynamics between bacteria and lytic phages, where two models for antagonistic coevolution dominate: arms-race dynamics (ARD) and fluctuating-selection dynamics (FSD). Here, we tested the ability for Pseudomonas aeruginosa to coevolve with phage OMKO1 during 10 passages in the laboratory, whether ARD versus FSD coevolution occurred, and how coevolution affected a predicted phenotypic trade-off between phage resistance and antibiotic sensitivity. We used a unique "deep" sampling design, where 96 bacterial clones per passage were obtained from the three replicate coevolving communities. Next, we examined phenotypic changes in growth ability, susceptibility to phage infection and resistance to antibiotics. Results confirmed that the bacteria and phages coexisted throughout the study with one community undergoing ARD, whereas the other two showed evidence for FSD. Surprisingly, only the ARD bacteria demonstrated the anticipated trade-off. Whole genome sequencing revealed that treatment populations of bacteria accrued more de novo mutations, relative to a control bacterial population. Additionally, coevolved bacteria presented mutations in genes for biosynthesis of flagella, type-IV pilus and lipopolysaccharide, with three mutations fixing contemporaneously with the occurrence of the phenotypic trade-off in the ARD-coevolved bacteria. Our study demonstrates that both ARD and FSD coevolution outcomes are possible in a single interacting bacteria-phage system and that occurrence of predicted phage-driven evolutionary trade-offs may depend on the genetics underlying evolution of phage resistance in bacteria. These results are relevant for the ongoing development of lytic phages, such as OMKO1, in personalized treatment of human patients, as an alternative to antibiotics.

Identification of additional fire blight resistance candidate genes following MinION Oxford Nanopore sequencing and assembly of BAC clone spanning the Malus fusca resistance locus

AbstractPyramiding different fire blight resistance genes and QTLs in future apple cultivars is the most eco-friendly way to combat this disease. Identification of strong fire blight resistance donors, and introgression of their resistance into apple breeding material are a continuing effort of breeding programs. Thus, enormous effort is been put into breeding research to understand host – pathogen interactions and mechanisms of resistance found in Malus. The crabapple Malus fusca (accession MAL0045) is highly resistant to fire blight, and although resistance is strain-dependent, resistance of MAL0045 is not overcome by any known strain of Erwinia amylovora to date. A strong fire blight resistance locus (FB_Mfu10) was fine mapped to an interval of 0.33 Centimorgan (cM) on linkage group (LG) 10 of MAL0045 using 1888 progenies. Subsequently, a single bacterial artificial chromosome (BAC) clone (46H22), which harbours FB_Mfu10-resistance alleles, was identified in a MAL0045 BAC library and sequenced using MiSeq illumina leading to the assembly of 45 contigs. Analyses of the sequence of 46H22 led to the identification of a receptor-like kinase candidate gene. Here, we report about resequencing 46H22 using MinION Oxford Nanopore and successfully assembled the sequences into a single contig, which allowed for identifying additional candidate genes.

Molecular Analyses of Clinical Isolates and Recombinant SARS-CoV-2 Carrying B.1 and B.1.617.2 Spike Mutations Suggest a Potential Role of Non-Spike Mutations in Infection Kinetics.

Some of the emerging severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants are less susceptible to neutralization with post-vaccine sera and monoclonal antibodies targeting the viral spike glycoprotein. This raises concerns of disease control, transmissibility, and severity. Numerous substitutions have been identified to increase viral fitness within the nucleocapsid and nonstructural proteins, in addition to spike mutations. Therefore, we sought to generate infectious viruses carrying only the variant-specific spike mutations in an identical backbone to evaluate the impact of spike and non-spike mutations in the virus life cycle. We used en passant mutagenesis to generate recombinant viruses carrying spike mutations of B.1 and B.1.617.2 variants using SARS-CoV-2- bacterial artificial chromosome (BAC). Neutralization assays using clinical sera yielded comparable results between recombinant viruses and corresponding clinical isolates. Non-spike mutations for both variants neither seemed to effect neutralization efficiencies with monoclonal antibodies nor the response to treatment with inhibitors. However, live-cell imaging and microscopy revealed differences, such as persisting syncytia and pronounced cytopathic effect formation, as well as their progression between BAC-derived viruses and clinical isolates in human lung epithelial cell lines and primary bronchial epithelial cells. Complementary RNA analyses further suggested a potential role of non-spike mutations in infection kinetics.

Construction of a Novel Infectious Clone of Recombinant Herpesvirus of Turkey Fc-126 Expressing VP2 of IBDV.

The increased virulence of infectious bursal disease virus (IBDV) is a threat to the chicken industry. The construction of novel herpesvirus of turkey-vectored (HVT) vaccines expressing VP2 of virulent IBDV may be a promising vaccine candidate for controlling this serious disease in chickens. We generated a novel infectious clone of HVT Fc-126 by inserting mini-F sequences in lieu of the glycoprotein C (gC) gene. Based on this bacterial artificial chromosome (BAC), a VP2 expression cassette containing the pMCMV IE promoter and a VP2 sequence from the virulent IBDV NJ09 strain was inserted into the noncoding area between the UL55 and UL56 genes to generate the HVT vector VP2 recombinant, named HVT-VP2-09. The recovered vectored mutant HVT-VP2-09 exhibited higher titers (p = 0.0202 at 36 h) or similar growth kinetics to the parental virus HVT Fc-126 (p = 0.1181 at 48 h and p = 0.1296 at 64 h). The high reactivation ability and strong expression of VP2 by HVT-VP2-09 in chicken embryo fibroblasts (CEFs) were confirmed by indirect immunofluorescence (IFA) and Western blotting. The AGP antibodies against IBDV were detected beginning at 3 weeks post-inoculation (P.I.) of HVT-VP2-09 in 1-day-old SPF chickens. Seven of ten chickens immunized with HVT-VP2-09 were protected post-challenge (P.C.) with the virulent IBDV NJ09 strain. In contrast, all chickens in the challenge control group showed typical IBD lesions in bursals, and eight of ten died P.C. In this study, we demonstrated that (i) a novel HVT BAC with the whole genome of the Fc-126 strain was obtained with the insertion of mini-F sequences in lieu of the gC gene; (ii) HVT-VP2-09 harboring the VP2 expression cassette from virulent IBDV exhibited in vitro growth properties similar to those of the parental HVT virus in CEF cells; and (iii) HVT-VP2-09 can provide efficient protection against the IBDV NJ09 strain.

Evaluation of safety and immunogenicity of duck-plague virus gC/gE double gene deletion.

Duck plague caused by duck plague virus (DPV) is a highly contagious disease that can cause serious morbidity and death in waterfowl such as ducks and geese, and bring huge economic losses to the duck industry. In this study, on the basis of the duck plague virus gC gene deletion strain CHv-ΔgC, based on the duck plague virus bacterial artificial chromosome (BAC) platform in our laboratory, the gE gene was knocked out using the traceless deletion technology to obtain gC/gE double gene deletion candidate vaccine strain CHv-ΔgC/gE. The double gene deletion strain (CHv-ΔgC/gE) constructed in this study has greatly weakened virulence, no pathogenicity to ducks, and stable genetic characteristics in vitro and in vivo. Ducks immunized with CHv-ΔgC/gE can produce neutralizing antibodies and ELISA antibody levels comparable to those of commercial duck plague attenuated vaccine immunization, and can resist 100 LD50 CHv challenge of ducks, with good immune protection effect. It has the potential to be further developed into duck plague gC/gE double gene deletion, marked attenuated vaccine.

Aberrant brain functional and structural developments in MECP2 duplication rats

Transgenic animal models with homologous etiology provide a promising way to pursue the neurobiological substrates of the behavioral deficits in autism spectrum disorder (ASD). Gain-of-function mutations of MECP2 cause MECP2 duplication syndrome, a severe neurological disorder with core symptoms of ASD. However, abnormal brain developments underlying the autistic-like behavioral deficits of MECP2 duplication syndrome are rarely investigated. To this end, a human MECP2 duplication (MECP2-DP) rat model was created by the bacterial artificial chromosome transgenic method. Functional and structural magnetic resonance imaging (MRI) with high-field were performed on 16 male MECP2-DP rats and 15 male wildtype rats at postnatal 28 days, 42 days, and 56 days old. Multimodal fusion analyses guided by locomotor-relevant metrics and social novelty time separately were applied to identify abnormal brain networks associated with diverse behavioral deficits induced by MECP2 duplication. Aberrant functional developments of a core network primarily composed of the dorsal medial prefrontal cortex (dmPFC) and retrosplenial cortex (RSP) were detected to associate with diverse behavioral phenotypes in MECP2-DP rats. Altered developments of gray matter volume were detected in the hippocampus and thalamus. We conclude that gain-of-function mutations of MECP2 induce aberrant functional activities in the default-mode-like network and aberrant volumetric changes in the brain, resulting in autistic-like behavioral deficits. Our results gain critical insights into the biomarker of MECP2 duplication syndrome and the neurobiological underpinnings of the behavioral deficits in ASD.

A synthetic DNA template for fast manufacturing of versatile single epitope mRNA

A flexible, affordable, and rapid vaccine platform is necessary to unlock the potential of personalized cancer vaccines in order to achieve full clinical efficiency. mRNA cancer vaccine manufacture relies on the rigid sequence design of multiepitope constructs produced by laborious bacterial cloning and time-consuming plasmid preparation. Here, we introduce a synthetic DNA template (SDT) assembly process, which allows cost- and time-efficient manufacturing of single (neo)epitope mRNA. We benchmarked SDT-derived mRNA against mRNA derived from a plasmid DNA template (PDT), showing that monocyte-derived dendritic cells (moDCs) electroporated with SDT-mRNA or PDT-mRNA, encoding HLA-I- or HLA-II-restricted (neo)epitopes, equally activated T cells that were modified to express the cognate T cell receptors. Furthermore, we validated the SDT-mRNA platform for neoepitope immunogenicity screening using the characterized HLA-A2-restricted neoepitope DHX40B and four new candidate HLA-A2-restricted melanoma neoepitopes. Finally, we compared SDT-mRNA with PDT-mRNA for vaccine development purposes. moDCs electroporated with mRNA encoding the HLA-A2-restricted, mutated Melan-A/Mart-1 epitope together with TriMix mRNA-generated high levels of functional Melan-A/Mart-1-specific CD8+ T cells. In conclusion, SDT single epitope mRNA can be manufactured in a more flexible, cost-efficient, and time-efficient way compared with PDT-mRNA, allowing prompt neoepitope immunogenicity screening, and might be exploited for the development of personalized cancer vaccines.

Multi-epitope chimeric vaccine designing and novel drug targets prioritization against multi-drug resistant Staphylococcus pseudintermedius

Biofilm synthesizing multi-drug resistant Staphylococcus pseudintermedius bacteria has been recognized as the human infectious agent. It has been detected in the diseases of skin, ear, and postoperative infections. Its infections are becoming a major health problem due to its multi-drug resistance capabilities. However, no commercial vaccine for the treatment of its infections is currently available in the market. Here we employed the subtractive proteomics and reverse vaccinology approach to determine the potential novel drug and vaccine targets against S. pseudintermedius infections in humans. After screening the core-proteome of the 39 complete genomes of S. pseudintermedius, 2 metabolic pathways dependent and 34 independent proteins were determined as novel potential drug targets. Two proteins were found and used as potential candidates for designing the chimeric vaccine constructs. Depending on the properties such as antigenicity, toxicity and solubility, multi-epitope based vaccines constructs were designed. For immunogenicity enhancement, different specific sequences like linkers, PADRE sequences and molecular adjuvants were added. Molecular docking and molecular dynamic simulation analyses were performed to evaluate the prioritized vaccine construct’s interactions with human immune cells HLA and TLR4. Finally, the cloning and expression ability of the vaccine construct was determined in the bacterial cloning system and human body immune response was predicted through immune simulation analysis. In conclusion, this study proposed the potential drug and vaccine targets and also designed a chimera vaccine to be tested and validated against infectious S. pseudintermedius species.

Analysis of decomposing wood wastes in the Lagos Lagoon using terminal-restriction fragment length polymorphism and illumina sequencing platform

Indiscriminate disposal of wood shavings and sawdust into the Lagos lagoon usually constitute environmental hazard and varying degree of threats to marine biotic communities. In this study we applied terminal-restriction fragment length polymorphism (T-RFLP) and illumina sequencing of 16S ribosomal RNA gene, to describe the microbial ecology of decomposing wood wastes in the Lagos lagoon, Nigeria. The terminal restriction fragments (TRFs) generated corresponds to over 100 bacterial genera and numerous uncultured bacterial clones. Phylogenetic analysis of the TRFs of 16S rRNA gene sequences and cultured bacterial sequences established genetic relatedness and divergent relationships. T-RFLP in combination with illumina sequencing platform identified bacterial species (Acinetobacter sp., Clostridium sp., Planctomyces sp., Escherichia coli, Pantoea agglomerans, Dyella japonica, Ochrobactrum sp., Prevotella sp., Runella slithyformis, Enterobacter aerogenes, Gordonia amicalis, Klebsiella sp., Thalassospira sp. and Serratia sp.) not previously accounted for in culture-dependent analysis. Bacterial strains such as Ruminococcus flavefaciens, Fibrobacter succinogenes, Simiduia agarivorans, Microbulbifer sp. and Cellulomonas fimi further identified in illumina sequences were found to be involved in the biodegradation of lignin and lignin derivatives. Hence, understanding of the diverse autochthonous microbiota of decomposing wood wastes in the Lagos lagoon is essential in the development of effective biotechnology programme, to tackling the menace of sawdust pollution.

Susceptibility of Staphylococcus aureus strains to commercial therapeutic phage preparations.

The aim of this study was to determine the susceptibility of Staphylococcus aureus strains to commercial phage preparations. The strains were isolated from clinical patients as well as from nasal mucosa of healthy carriers. The elevating number of antibiotic-resistant S. aureus strains present a therapeutic challenge, especially in high-risk patients. One of the promising ways to solve this problem is phage therapy. Susceptibility of 111 carrier strains of S.aureus (4 strains were methicillin-resistant; MRSA) and 81 clinical isolates from bloodstream or skin and soft tissue infections (28 were MRSA) to four commercial phage preparations was assessed in vitro by spot assay. The clonality of S. aureus strains was determined by spa typing. Spa typing revealed 97 distinct spa types. A proportion of 73-80 % of the tested S. aureus strains were revealed to have in vitro phage susceptibility, depending on the clonal affiliation of the strains and phage preparation tested. The susceptibility to phage preparations was significantly higher in MRSA strains (p < 0.001). In vitro results indicate a promising therapeutic potential of the tested commercial anti-staphylococcal phage preparations. They could be applied to a broad spectrum of bacterial clones, and have an excellent activity especially against MRSA strains (Tab. 2, Fig. 2, Ref. 43).

Self-Repairing Herpesvirus Saimiri Deletion Variants.

Herpesvirus saimiri (HVS) is discussed as a possible vector in gene therapy. In order to create a self-repairing HVS vector, the F plasmid vector moiety of the bacterial artificial chromosome (BAC) was transposed via Red recombination into the virus genes ORF22 or ORF29b, both important for virus replication. Repetitive sequences were additionally inserted, allowing the removal of the F-derived sequences from the viral DNA genome upon reconstitution in permissive epithelial cells. Moreover, these self-repair-enabled BACs were used to generate deletion variants of the transforming strain C488 in order to minimalize the virus genome. Using the en passant mutagenesis with two subsequent homologous recombination steps, the BAC was seamlessly manipulated. To ensure the replication capacity in permissive monkey cells, replication kinetics for all generated virus variants were documented. HVS variants with increased insert capacity reached the self-repair within two to three passages in permissive epithelial cells. The seamless deletion of ORFs 3/21, 12–14, 16 or 71 did not abolish replication competence. Apoptosis induction did not seem to be altered in human T cells transformed with deletion variants lacking ORF16 or ORF71. These virus variants form an important step towards creating a potential minimal virus vector for gene therapy, for example, in human T cells.

Differential Cellular Balance of Olfactory and Vomeronasal Epithelia in a Transgenic BACHD Rat Model of Huntington's Disease.

Background. For neurodegenerative diseases such as Huntington’s disease (HD), early diagnosis is essential to treat patients and delay symptoms. Impaired olfaction, as observed as an early symptom in Parkinson´s disease, may also constitute a key symptom in HD. However, there are few reports on olfactory deficits in HD. Therefore, we aimed to investigate, in a transgenic rat model of HD: (1) whether general olfactory impairment exists and (2) whether there are disease-specific dynamics of olfactory dysfunction when the vomeronasal (VNE) and main olfactory epithelium (MOE) are compared. Methods. We used male rats of transgenic line 22 (TG22) of the bacterial artificial chromosome Huntington disease model (BACHD), aged 3 days or 6 months. Cell proliferation, apoptosis and macrophage activity were examined with immunohistochemistry in the VNE and MOE. Results. No differences were observed in cellular parameters in the VNE between the groups. However, the MOE of the 6-month-old HD animals showed a significantly increased number of mature olfactory receptor neurons. Other cellular parameters were not affected. Conclusions. The results obtained in the TG22 line suggest a relative stability in the VNE, whereas the MOE seems at least temporarily affected.

Development of a humanized HLA-A30 transgenic mouse model.

BackgroundThere are remarkable genetic differences between animal major histocompatibility complex (MHC) systems and the human leukocyte antigen (HLA) system. HLA transgenic humanized mouse model systems offer a much better method to study the HLA‐A‐related principal mechanisms for vaccine development and HLA‐A‐restricted responses against infection in human.MethodsA recombinant gene encoding the chimeric HLA‐A30 monochain was constructed. This HHD molecule contains the following: α1‐α2 domains of HLA‐A30, α3 and cytoplasmic domains of H‐2Db, linked at its N‐terminus to the C‐terminus of human β2m by a 15‐amino‐acid peptide linker. The recombinant gene encoding the chimeric HLA‐A30 monochain cassette was introduced into bacterial artificial chromosome (BAC) CH502‐67J3 containing the HLA‐A01 gene locus by Red‐mediated homologous recombination. Modified BAC CH502‐67J3 was microinjected into the pronuclei of wild‐type mouse oocytes. This humanized mouse model was further used to assess the immune responses against influenza A virus (H1N1) pdm09 clinically isolated from human patients. Immune cell population, cytokine production, and histopathology in the lung were analyzed.ResultsWe describe a novel human β2m‐HLA‐A30 (α1α2)‐H‐2Db (α3 transmembrane cytoplasmic) (HHD) monochain transgenic mouse strain, which contains the intact HLA‐A01 gene locus including 49 kb 5′‐UTR and 74 kb 3′‐UTR of HLA‐A01*01. Five transgenic lines integrated into the large genomic region of HLA‐A gene locus were obtained, and the robust expression of exogenous transgene was detected in various tissues from A30‐18# and A30‐19# lines encompassing the intact flanking sequences. Flow cytometry revealed that the introduction of a large genomic region in HLA‐A gene locus can influence the immune cell constitution in humanized mice. Pdm09 infection caused a similar immune response among HLA‐A30 Tg humanized mice and wild‐type mice, and induced the rapid increase of cytokines, including IFN‐γ, TNF‐α, and IL‐6, in both HLA‐A30 humanized Tg mice and wild‐type mice. The expression of HLA‐A30 transgene was dramatically promoted in tissues from A30‐9# line at 3 days post‐infection (dpi).ConclusionsWe established a promising preclinical research animal model of HLA‐A30 Tg humanized mouse, which could accelerate the identification of novel HLA‐A30‐restricted epitopes and vaccine development, and support the study of HLA‐A‐restricted responses against infection in humans.

Identification of sex-specific markers and ZW-chromosome DNA clones from the genomic BAC library of the Chinese mitten crab Eriocheir sinensis

A ZW-ZZ sex determination system was previously proposed based on sex linkage analysis in the Chinese mitten crab, Eriocheir sinensis. In this study, we successfully identified a sex-specific marker from sex-linked markers in the genetic map and verified in different wild populations of E. sinensis. To further identify ZW chromosome DNA, we constructed a highly redundant genomic bacterial artificial chromosome (BAC) library for the Chinese mitten crab. The library was generated from high-molecular weight genomic DNA isolated from hemocytes and muscle in a female crab, which was comprising of 153,984 clones arrayed in 401,384-well microtiter plates. The average inserts size is 72 kb, representing 8.7-fold haploid genome coverage. By using sex specific markers, PCR-based screening was performed to identify ZW-derived BAC clones. Three W and Z-derived BAC clones were isolated, respectively. One of W-derived (P282E18) BAC clones was sequenced and assembled into a 43 kb contig without any gap. The W-derived BAC inserts can be mapped to an assembled contig of E. sinensis genome with 95% similarity in the aligned region, demonstrating that the BAC library is a valuable resource for discriminating Z and W chromosome contigs in the assembled haploid genome. The sex-specific marker provides a powerful tool not only for distinguishing the gender of larval or early juvenile E. sinensis without sexual dimorphism and sex-reversed individuals in monosex crab breeding, but also for identification of ZW-derived BAC clones used in the assembly of Z and W chromosomes in E. sinensis.

Engineering and Characterization of Avian Coronavirus Mutants Expressing Fluorescent Reporter Proteins from the Replicase Gene.

Infectious bronchitis virus (IBV) is an avian coronavirus that causes infectious bronchitis, an acute and highly contagious respiratory disease of chickens. IBV evolution under the pressure of comprehensive and widespread vaccination requires surveillance for vaccine resistance, as well as periodic vaccine updates. Reverse genetics systems are very valuable tools in virology, as they facilitate rapid genetic manipulation of viral genomes, thereby advancing basic and applied research. We report here the construction of an infectious clone of IBV strain Beaudette as a bacterial artificial chromosome (BAC). The engineered full-length IBV clone allowed the rescue of an infectious virus that was phenotypically indistinguishable from the parental virus. We used the infectious IBV clone and examined whether an enhanced green fluorescent protein (EGFP) can be produced by the replicase gene ORF1 and autocatalytically released from the replicase polyprotein through cleavage by the main coronavirus protease. We show that IBV tolerates insertion of the EGFP ORF at the 3' end of the replicase gene, between the sequences encoding nsp13 and nsp16 (helicase, RNA exonuclease, RNA endonuclease, and RNA methyltransferase). We further show that EGFP is efficiently cleaved from the replicase polyprotein and can be localized in double-membrane vesicles along with viral RNA polymerase and double-stranded RNA, an intermediate of IBV genome replication. One of the engineered reporter EGFP viruses were genetically stable during passage in cultured cells. We demonstrate that the reporter EGFP viruses can be used to study virus replication in host cells and for antiviral drug discovery and development of diagnostic assays. IMPORTANCE Reverse genetics systems based on bacterial artificial chromosomes (BACs) are the most valuable systems in coronavirus research. Here, we describe the establishment of a reverse genetics system for the avian coronavirus strain Beaudette, the most intensively studied strain. We cloned a copy of the avian coronavirus genome into a BAC vector and recovered infectious virus in permissive cells. We used the new system to construct reporter viruses that produce enhanced green fluorescent protein (EGFP). The EGFP coding sequence was inserted into 11 known cleavage sites of the major coronavirus protease in the replicase gene ORF1. Avian coronavirus tolerated the insertion of the EGFP coding sequence at three sites. The engineered reporter viruses replicated with parental efficiency in cultured cells and were sufficiently genetically stable. The new system facilitates functional genomics of the avian coronavirus genome but can also be used for the development of novel vaccines and anticoronaviral drugs.