A novel and accurate full-length HTT mouse model for Huntington’s disease

Here, we report the generation and characterization of a novel Huntington’s disease (HD) mouse model BAC226Q by using a bacterial artificial chromosome (BAC) system, expressing full-length human HTT with ~226 CAG-CAA repeats and containing endogenous human HTT promoter and regulatory elements. BAC226Q recapitulated a full-spectrum of age-dependent and progressive HD-like phenotypes without unwanted and erroneous phenotypes. BAC226Q mice developed normally, and gradually exhibited HD-like psychiatric and cognitive phenotypes at 2 months. From 3 to 4 months, BAC226Q mice showed robust progressive motor deficits. At 11 months, BAC226Q mice showed significant reduced life span, gradual weight loss and exhibited neuropathology including significant brain atrophy specific to striatum and cortex, striatal neuronal death, widespread huntingtin inclusions, and reactive pathology. Therefore, the novel BAC226Q mouse accurately recapitulating robust, age-dependent, progressive HD-like phenotypes will be a valuable tool for studying disease mechanisms, identifying biomarkers, and testing gene-targeting therapeutic approaches for HD.

Engineering, decoding and systems-level characterization of chimpanzee cytomegalovirus

The chimpanzee cytomegalovirus (CCMV) is the closest relative of human CMV (HCMV). Because of the high conservation between these two species and the ability of human cells to fully support CCMV replication, CCMV holds great potential as a model system for HCMV. To make the CCMV genome available for precise and rapid gene manipulation techniques, we captured the genomic DNA of CCMV strain Heberling as a bacterial artificial chromosome (BAC). Selected BAC clones were reconstituted to infectious viruses, growing to similar high titers as parental CCMV. DNA sequencing confirmed the integrity of our clones and led to the identification of two polymorphic loci and a deletion-prone region within the CCMV genome. To re-evaluate the CCMV coding potential, we analyzed the viral transcriptome and proteome and identified several novel ORFs, splice variants, and regulatory RNAs. We further characterized the dynamics of CCMV gene expression and found that viral proteins cluster into five distinct temporal classes. In addition, our datasets revealed that the host response to CCMV infection and the de-regulation of cellular pathways are in line with known hallmarks of HCMV infection. In a first functional experiment, we investigated a proposed frameshift mutation in UL128 that was suspected to restrict CCMV’s cell tropism. In fact, repair of this frameshift re-established productive CCMV infection in endothelial and epithelial cells, expanding the options of CCMV as an infection model. Thus, BAC-cloned CCMV can serve as a powerful tool for systematic approaches in comparative functional genomics, exploiting the close phylogenetic relationship between CCMV and HCMV.

HuluFISH non-denaturing in situ detection of genomic DNA opened by CRISPR-Cas9 Nickase and Exonuclease

DNA fluorescence in situ hybridization (FISH) has been widely used in diagnosis and genetic research. Traditional Bacterial artificial chromosome (BAC) or oligonucleotide based probe to detect DNA in situ is only effective when the target is relatively large, usually over 150Kb DNA fragments. And it involves heat denaturation steps to open the DNA for in situ hybridization. The heat process can affect the fine structure of nuclei. Here we reported a novel method based on Cas9 nickase and exonuclease digestion of double strand DNA and permanently mark the DNA in single strand state for FISH. With this novel design, we detected non-repetitive genomic loci as small as 2Kb.

The LORF5 Gene Is Non-essential for Replication but Important for Duck Plague Virus Cell-to-Cell Spread Efficiently in Host Cells

Duck plague virus (DPV) can cause high morbidity and mortality in many waterfowl species within the order Anseriformes. The DPV genome contains 78 open reading frames (ORFs), among which the LORF2, LORF3, LORF4, LORF5, and SORF3 genes are unique genes of avian herpesvirus. In this study, to investigate the role of this unique LORF5 gene in DPV proliferation, we generated a recombinant virus that lacks the LORF5 gene by a two-step red recombination system, which cloned the DPV Chinese virulent strain (DPV CHv) genome into a bacterial artificial chromosome (DPV CHv-BAC); the proliferation law of LORF5-deleted mutant virus on DEF cells and the effect of LORF5 gene on the life cycle stages of DPV compared with the parent strain were tested. Our data revealed that the LORF5 gene contributes to the cell-to-cell transmission of DPV but is not relevant to virus invasion, replication, assembly, and release formation. Taken together, this study sheds light on the role of the avian herpesvirus-specific gene LORF5 in the DPV proliferation life cycle. These findings lay the foundation for in-depth functional studies of the LORF5 gene in DPV or other avian herpesviruses.

Variation and Evolution of Human Centromeres: A Field Guide and Perspective

We are entering a new era in genomics where entire centromeric regions are accurately represented in human reference assemblies. Access to these high-resolution maps will enable new surveys of sequence and epigenetic variation in the population and offer new insight into satellite array genomics and centromere function. Here, we focus on the sequence organization and evolution of alpha satellites, which are credited as the genetic and genomic definition of human centromeres due to their interaction with inner kinetochore proteins and their importance in the development of human artificial chromosome assays. We provide an overview of alpha satellite repeat structure and array organization in the context of these high-quality reference data sets; discuss the emergence of variation-based surveys; and provide perspective on the role of this new source of genetic and epigenetic variation in the context of chromosome biology, genome instability, and human disease.

Marfan Syndrome Caused by Disruption of the FBN1 Gene due to A Reciprocal Chromosome Translocation

Marfan syndrome (MFS) is a hereditary connective tissue disease caused by heterozygous mutations in the fibrillin-1 gene (FBN1) located on chromosome 15q21.1. A complex chromosomal rearrangement leading to MFS has only been reported in one case so far. We report on a mother and daughter with marfanoid habitus and no pathogenic variant in the FBN1 gene after next generation sequencing (NGS) analysis, both showing a cytogenetically reciprocal balanced translocation between chromosomes 2 and 15. By means of fluorescence in situ hybridization of Bacterial artificial chromosome (BAC) clones from the breakpoint area on chromosome 15 the breakpoint was narrowed down to a region of approximately 110 kb in FBN1. With the help of optical genome mapping (OGM), the translocation breakpoints were further refined on chromosomes 2 and 15. Sequencing of the regions affected by the translocation identified the breakpoint of chromosome 2 as well as the breakpoint of chromosome 15 in the FBN1 gene leading to its disruption. To our knowledge, this is the first report of patients with typical clinical features of MFS showing a cytogenetically reciprocal translocation involving the FBN1 gene. Our case highlights the importance of structural genome variants as an underlying cause of monogenic diseases and the useful clinical application of OGM in the elucidation of structural variants.

TWIK-1 BAC-GFP Transgenic Mice, an Animal Model for TWIK-1 Expression

TWIK-1 is the first identified member of the two-pore domain potassium (K2P) channels that are involved in neuronal excitability and astrocytic passive conductance in the brain. Despite the physiological roles of TWIK-1, there is still a lack of information on the basic expression patterns of TWIK-1 proteins in the brain. Here, using a modified bacterial artificial chromosome (BAC), we generated a transgenic mouse (Tg mouse) line expressing green fluorescent protein (GFP) under the control of the TWIK-1 promoter (TWIK-1 BAC-GFP Tg mice). We confirmed that nearly all GFP-producing cells co-expressed endogenous TWIK-1 in the brain of TWIK-1 BAC-GFP Tg mice. GFP signals were highly expressed in various brain areas, including the dentate gyrus (DG), lateral entorhinal cortex (LEC), and cerebellum (Cb). In addition, we found that GFP signals were highly expressed in immature granule cells in the DG. Finally, our TWIK-1 BAC-GFP Tg mice mimic the upregulation of TWIK-1 mRNA expression in the hippocampus following the injection of kainic acid (KA). Our data clearly showed that TWIK-1 BAC-GFP Tg mice are a useful animal model for studying the mechanisms regulating TWIK-1 gene expression and the physiological roles of TWIK-1 channels in the brain.

Construction of stable mouse artificial chromosome from native mouse chromosome 10 for generation of transchromosomic mice

Mammalian artificial chromosomes derived from native chromosomes have been applied to biomedical research and development by generating cell sources and transchromosomic (Tc) animals. Human artificial chromosome (HAC) is a precedent chromosomal vector which achieved generation of valuable humanized animal models for fully human antibody production and human pharmacokinetics. While humanized Tc animals created by HAC vector have attained significant contributions, there was a potential issue to be addressed regarding stability in mouse tissues, especially highly proliferating hematopoietic cells. Mouse artificial chromosome (MAC) vectors derived from native mouse chromosome 11 demonstrated improved stability, and they were utilized for humanized Tc mouse production as a standard vector. In mouse, however, stability of MAC vector derived from native mouse chromosome other than mouse chromosome 11 remains to be evaluated. To clarify the potential of mouse centromeres in the additional chromosomes, we constructed a new MAC vector from native mouse chromosome 10 to evaluate the stability in Tc mice. The new MAC vector was transmitted through germline and stably maintained in the mouse tissues without any apparent abnormalities. Through this study, the potential of additional mouse centromere was demonstrated for Tc mouse production, and new MAC is expected to be used for various applications.

Construction, Characterization and Application of Recombinant Porcine Deltacoronavirus Expressing Nanoluciferase

Porcine deltacoronavirus (PDCoV), an emerging enteropathogenic coronavirus, causes diarrhoea in suckling piglets and has the potential for cross-species transmission. No effective PDCoV vaccines or antiviral drugs are currently available. Here, we successfully generated an infectious clone of PDCoV strain CHN-HN-2014 using a combination of bacterial artificial chromosome (BAC)-based reverse genetics system with a one-step homologous recombination. The recued virus (rCHN-HN-2014) possesses similar growth characteristics to the parental virus in vitro. Based on the established infectious clone and CRISPR/Cas9 technology, a PDCoV reporter virus expressing nanoluciferase (Nluc) was constructed by replacing the NS6 gene. Using two drugs, lycorine and resveratrol, we found that the Nluc reporter virus exhibited high sensibility and easy quantification to rapid antiviral screening. We further used the Nluc reporter virus to test the susceptibility of different cell lines to PDCoV and found that cell lines derived from various host species, including human, swine, cattle and monkey enables PDCoV replication, broadening our understanding of the PDCoV cell tropism range. Taken together, our reporter viruses are available to high throughput screening for antiviral drugs and uncover the infectivity of PDCoV in various cells, which will accelerate our understanding of PDCoV.