Bacterial Artificial Chromosome Construct Research Articles

Single Nucleotide Polymorphism-based Identification of Bacterial Artificial Chromosome-mediated Homologous Recombination.

Bacterial Artificial chromosome (BAC) recombineering is a powerful genetic manipulation tool for the efficient development of recombinant genetic resources. Long homology arms of more than 150 kb composed of BAC constructs not only substantially enhance genetic recombination events, but also provide a variety of single nucleotide polymorphisms (SNPs) that are useful markers for accurately docking BAC constructs at target sites. Even if the BAC construct is homologous to the sequences of the target region, different variations may be distributed between various SNPs within the region and those within the BAC construct. Once the BAC construct carrying these variations was precisely replaced in the target region, the SNP profiles within the target genomic locus were directly replaced with those in the BAC. This alteration in SNP profiles ensured that the BAC construct accurately targeted the designated site. In this study, we introduced restriction fragment length polymorphism or single-strand conformation polymorphism analyses to validate and evaluate BAC recombination based on changes in SNP patterns. These methods provide a simple and economical solution to validation steps that can be cumbersome with large homologous sequences, facilitating access to the production of therapeutic resources or disease models based on BAC-mediated homologous recombination.

Attenuation of equine herpesvirus 1 through deletion of gE gene and its pathological evaluation in murine model.

Equine herpesvirus 1 (EHV1) infection is aglobal health problem in equines and the virus is responsible for abortions, respiratory disease and myeloencephalitis in horses. Disease management requires proper biosecurity and immunoprophylactic measures. Vaccines strengthening both arms of immunity are essential for proper control and there has been acontinuous focus in this area for generation of better vaccines. Here we report construction of bacterial artificial chromosome (BAC) clone of EHV-1 strain Tohana for mutagenesis of the virus and generation of gE gene deletion mutant EHV1. The BAC clone was generated by inserting the mini-F plasmid replacing ORF71 of EHV1 and transforming into E. coli for generation of EHV1-BAC. The infectious virus was regenerated from EHV-1 BAC DNA in RK13 cells. To check utility of EHV1-BAC, we have generated mutant EHV1 by deleting the virulence-associated gE gene. The mutant virus (vToHΔgE) showed significantly reduced plaque size without affecting replication efficiency. Pathological evaluation of lesions in BALB/c mice infected with vToHΔgE revealed reduction in clinical signs and pathology in comparison to the wild-type virus. Generation of infectious BAC of EHV1 and its usage in construction of attenuated viruses shows potential of the technology for development of indigenous modified live vaccine for EHV1. Keywords: quine herpesvirus 1; bacterial artificial chromosome (BAC); mutation; glycoprotein E; vaccine.

Abstract 2584: Development of an Fc gamma receptor fully-humanized mouse model to support the accurate preclinical study of therapeutic antibodies

Abstract The Fc gamma receptor (FcγR), a member of the immunoglobulin superfamily, binds the IgG Fc segment of antibodies and is mainly expressed on the surface of immune cell. The FcγR binds to the Fc region of an antibody that is attached to target cells, transmitting an activation or inhibition signal to the FcγR expressing cells, resulting in various biological responses and functions. Many therapeutic antibodies are developed based on the engineering of the Fc region to fine-tune the FcγR mediated effects. There are four subtypes of mouse FcγRs, including Fcgr1, Fcgr2b, Fcgr3, and Fcgr4. In contrast, human FcγRs consist of at least seven members, including FCGR1A, FCGR1B, FCGR2A, FCGR2B, FCGR2C, FCGR3A, and FCGR3B. Additionally, the expression pattern of even the orthologous FcγR genes varies between humans and mice. e.g., the distribution of mouse FCGR3 and FCGR2. Therefore, animals harboring the murine FcγRs are not ideal models for the accurate preclinical evaluation of human therapeutic antibodies. To address this problem, we established an FcγRs fully humanized mouse model by injecting an engineered human bacterial artificial chromosome construct carrying all seven human FcγR genes into mouse zygotes. The transgene carriers were mated with another strain in which all mouse FcγR genes were knocked out to establish the colony. Unlike the endogenous mouse FcγR genes expression profiles, the transgenic human FcγR genes regulated under the control of the human control elements retain the human expression pattern. For example, the transgenic human FCGR3A was observed on mouse NK cells and macrophages, whereas the FCGR3B was detected on mouse neutrophils only, which were consistent with their expression patterns in humans. Antibody-dependent cellular cytotoxicity (ADCC) experiment indicated that a human therapeutic antibody could direct NK cells derived from the FcγRs fully-humanized mice to kill target tumor cells. Moreover, a significant anti-tumor effect could also be achieved by an ADCC based human antibody in tumor-bearing FcγRs humanized mice. Taken together, the FcγR gene fully-humanized model established in this study demonstrates a human FcγR expression pattern and functionality, thus providing a valuable tool for accurate preclinical evaluation of human therapeutic antibodies. Citation Format: Mingkun Zhang, Cunxiang Ju, Ying Li, Yunlong Jiang, Weiwei Yu, Shuai Li, Hongyan Sun, Zhiying Li, Jing Zhao, Xiang Gao. Development of an Fc gamma receptor fully-humanized mouse model to support the accurate preclinical study of therapeutic antibodies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2584.

Role of the C9ORF72 Gene in the Pathogenesis of Amyotrophic Lateral Sclerosis and Frontotemporal Dementia.

Since the discovery of the C9ORF72 gene in 2011, great advances have been achieved in its genetics and in identifying its role in disease models and pathological mechanisms; it is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). ALS patients with C9ORF72 expansion show heterogeneous symptoms. Those who are C9ORF72 expansion carriers have shorter survival after disease onset than non-C9ORF72 expansion patients. Pathological and clinical features of C9ORF72 patients have been well mimicked via several models, including induced pluripotent stem cell-derived neurons and transgenic mice that were embedded with bacterial artificial chromosome construct and that overexpressing dipeptide repeat proteins. The mechanisms implicated in C9ORF72 pathology include DNA damage, changes of RNA metabolism, alteration of phase separation, and impairment of nucleocytoplasmic transport, which may underlie C9ORF72 expansion-related ALS/FTD and provide insight into non-C9ORF72 expansion-related ALS, FTD, and other neurodegenerative diseases.

Rapid Lentiviral Vector Producer Cell Line Generation Using a Single DNA Construct

Stable suspension producer cell lines for the production of vesicular stomatitis virus envelope glycoprotein (VSVg)-pseudotyped lentiviral vectors represent an attractive alternative to current widely used production methods based on transient transfection of adherent 293T cells with multiple plasmids. We report here a method to rapidly generate such producer cell lines from 293T cells by stable transfection of a single DNA construct encoding all lentiviral vector components. The resulting suspension cell lines yield titers as high as can be achieved with transient transfection, can be readily scaled up in single-use stirred-tank bioreactors, and are genetically and functionally stable in extended cell culture. By removing the requirement for efficient transient transfection during upstream processing of lentiviral vectors and switching to an inherently scalable suspension cell culture format, we believe that this approach will result in significantly higher batch yields than are possible with current manufacturing processes and enable better patient access to medicines based on lentiviral vectors.

Improved protocols for BAC insert DNA isolation, BAC end sequencing and FISH for construction of BAC based physical map of genes on the chromosomes.

Bacterial artificial chromosome (BAC) library is an important genomic resource useful in targeted marker development, positional cloning, physical mapping and a substrate for genome sequencing for better understanding the genome organization of a species. The present manuscript elucidates the improvement in protocols for economical and efficient BAC insert DNA isolation, BAC end sequencing and FISH for physical localization on the metaphase chromosome complements. BAC clones of Clarias magur, maintained in 384-well plate format in our laboratory, were used in this study. The protocols gave consistent and efficient results. We use routinely these protocols for BAC insert DNA extraction, generating end sequence data of the clone and constructing DNA probes to hybridize on the metaphase spreads of C. magur using FISH for physical their localization.

Development of a Sequence-Based Reference Physical Map of Pea (Pisum sativum L.).

Whole genome profiling (WGP) is a sequence-based physical mapping technology and uses sequence tags generated by next generation sequencing for construction of bacterial artificial chromosome (BAC) contigs of complex genomes. The physical map provides a framework for assembly of genome sequence and information for localization of genes that are difficult to find through positional cloning. To address the challenges of accurate assembly of the pea genome (∼4.2 GB of which approximately 85% is repetitive sequences), we have adopted the WGP technology for assembly of a pea BAC library. Multi-dimensional pooling of 295,680 BAC clones and sequencing the ends of restriction fragments of pooled DNA generated 1,814 million high quality reads, of which 825 million were deconvolutable to 1.11 million unique WGP sequence tags. These WGP tags were used to assemble 220,013 BACs into contigs. Assembly of the BAC clones using the modified Fingerprinted Contigs (FPC) program has resulted in 13,040 contigs, consisting of 213,719 BACs, and 6,294 singleton BACs. The average contig size is 0.33 Mbp and the N50 contig size is 0.62 Mbp. WGPTM technology has proved to provide a robust physical map of the pea genome, which would have been difficult to assemble using traditional restriction digestion based methods. This sequence-based physical map will be useful to assemble the genome sequence of pea. Additionally, the 1.1 million WGP tags will support efficient assignment of sequence scaffolds to the BAC clones, and thus an efficient sequencing of BAC pools with targeted genome regions of interest.

A novel mouse model of kidney fibrosis induced by glomerulus‐specific overexpression of the full‐length of human transforming growth factor‐β1 gene

Chronic renal disease is a common complication of several diseases including diabetes mellitus, hypertension and chronic glomerulonephritis. Approximately 10% of adults in developed countries suffer from chronic renal disease. Clinical progression of chronic renal disease leads to end‐stage renal disease that can be treated only with renal replacement therapy including dialysis and renal transplantation. There is currently no other therapeutic option for patients in terminal stages of the disease. The main reason of the scarce therapeutic strategies for this chronic and fatal disease is the lack of appropriate disease model to evaluate drug discovery. In the present study, we focused on the potent profibrotic activity of transforming growth factor‐β1 and its critical mechanistic role in glomerulosclerosis. We prepared and amplified a chimeric mouse podocin/human transforming growth factor‐β1 bacterial artificial chromosome construct, purified, linearized, separated by pulsed field gel electrophoresis and dialyzed before microinjection into fertilized eggs. We obtained several founders expressing the transgene as demonstrated by Southern blotting. As expected, the transgenic mice have increased plasma levels of human transforming growth factor‐β1, creatinine and blood urea nitrogen from 8‐weeks of age. The urine levels of fatty acid‐binding protein, a marker of kidney injury, and human transforming growth factor‐β1 were also significantly increased in the transgenic mice compared to wild type mice. Trichrome staining of the kidneys disclosed extensive deposition of extracellular matrix proteins in the transgenic mice compared to their wild type counterparts. In addition to highlighting the important role of transforming growth factor‐β1 in the pathogenesis of kidney fibrosis, the results of this study led to development of a novel mouse model of kidney fibrosis that may be useful for drug discovery.Support or Funding InformationI have no financial relationships to disclose.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Comparative cytogenetic analysis in Vigna sp. revealed by BAC-FISH

Vigna Savi species constitute a group of worldwide legumes of great socioeconomic importance. Progress in its genomic resources allowed the development of a consensus genetic map with a high-density of SNP markers and construction of Bacterial Artificial Chromosome (BAC) libraries. Fluorescent in situ hybridization (FISH) using BAC clones as probes (BAC-FISH) became a powerful tool for synteny and collinearity analyses among closely related species in several groups of plants. Previous work using BAC-FISH with probes of the P. vulgaris ‘BAT93’ library of V. unguiculata (Vu) and V. aconitifolia (Vac) chromosomes showed partial conservation of macrosynteny with chromosomal rearrangements. For a better understanding of the genomic organization and karyotype evolution in Vigna, we performed a comparative cytogenetic study with three Vigna species (2n = 22), using BAC-FISH. Four single-copy BACs of chromosome 3 and chromosome 11 of V. unguiculata (Vu3 and Vu11, respectively) were hybridized in situ on mitotic metaphase chromosomes of V. angularis (Van) and V. radiata (Vr). For chromosome 3, two Vu3 BACs (H31G07 and H50P11) were located in the same orientation at Vu, Van and Vr chromosomes, in interstitial regions in the short and long arms, respectively. These results suggest a conservation of synteny for both markers. On the other hand, H49E24 and H85I15 BACs were located respectively in terminal region in the short arm and subterminal region in the long arm of Vu11, while a distinct pattern was observed in Van, which showed both BACs at adjacent positions in the short arm. This suggests that a pericentric inversion involving BAC H85I15 occurred in one of these species. Based on the present results, associated with our previous works, the inversion observed on Vu11 seems to have occurred in V. unguiculata after Vigna and Phaseolus separation, since position of other BAC clones were different in Vu when compared to Vac, Van and Pv. These data demonstrate the feasibility of the BAC-FISH technique in comparative chromosome mapping, showing a break of macrosynteny among species of Phaseoloid clade. This is an initial step of an extensive macrosynteny study with BAC-FISH in Vigna.

Bacterial artificial chromosomes as recombinant reporter constructs to investigate gene expression and regulation in echinoderms.

Genome sequences contain all the necessary information-both coding and regulatory sequences-to construct an organism. The developmental process translates this genomic information into a three-dimensional form. One interpretation of this translation process can be described using gene regulatory network (GRN) models, which are maps of interactions among regulatory gene products in time and space. As high throughput investigations reveal increasing complexity within these GRNs, it becomes apparent that efficient methods are required to test the necessity and sufficiency of regulatory interactions. One of the most complete GRNs for early development has been described in the purple sea urchin, Strongylocentrotus purpuratus. This work has been facilitated by two resources: a well-annotated genome sequence and transgenes generated in bacterial artificial chromosome (BAC) constructs. BAC libraries played a central role in assembling the S. purpuratus genome sequence and continue to serve as platforms for generating reporter constructs for use in expression and regulatory analyses. Optically transparent echinoderm larvae are highly amenable to transgenic approaches and are therefore particularly well suited for experiments that rely on BAC-based reporter transgenes. Here, we discuss the experimental utility of BAC constructs in the context of understanding developmental processes in echinoderm embryos and larvae.

The PPAR-gamma-binding sequence Pal3 is necessary for basal but dispensable for high-fat diet regulated human renin expression in the kidney.

We reported earlier that PPAR-gamma regulates renin transcription through a human-specific atypical binding sequence termed hRen-Pal3. Here we developed a mouse model to investigate the functional relevance of the hRen-Pal3 sequence in vivo since it might be responsible for the increased renin production in obesity and thus for the development of accompanying arterial hypertension. We used bacterial artificial chromosome construct and co-placement strategy to generate two transgenic mouse lines expressing the human renin gene from identical genomic locus without affecting the intrinsic mouse renin expression. One line carried a wild-type hRen-Pal3 in the transgene (Pal3wt strain) and the other a mutated non-functional Pal3 (Pal3mut strain). Human renin expression was correctly targeted to the renin-producing juxtaglomerular (JG) cells of kidney in both lines. However, Pal3mut mice had lower basal human renin expression. Since human renin does not recognize mouse angiotensinogen as substrate, the blood pressure was not different between the strains. Stimulation of renin production with the angiotensin-converting enzyme inhibitor enalapril equipotentially stimulated the human renin expression in Pal3wt and Pal3mut mice. High-fat diet for 10weeks which is known to activate PPAR-gamma failed to increase human renin mRNA in kidneys of either strain. These findings showed that the human renin PPAR-gamma-binding sequence hRen-Pal3 is essential for basal renin expression but dispensable for the cell-specific and high-fat diet regulated renin expression in the kidney.

A Plasmid Set for Efficient Bacterial Artificial Chromosome (BAC) Transgenesis in Zebrafish

Transgenesis of large DNA constructs is essential for gene function analysis. Recently, Tol2 transposase-mediated transgenesis has emerged as a powerful tool to insert bacterial artificial chromosome (BAC) DNA constructs into the genome of zebrafish. For efficient transgenesis, the genomic DNA piece in the BAC construct needs to be flanked by Tol2 transposon sites, and the constructs should contain a transgenesis marker for easy identification of transgenic animals. We report a set of plasmids that contain targeting cassettes that allow the insertion of Tol2 sites and different transgenesis markers into BACs. Using BACs containing these targeting cassettes, we show that transgenesis is as efficient as iTol2, that preselecting for expression of the transgenesis marker increases the transgenesis rate, and that BAC transgenics faithfully recapitulate the endogenous gene expression patterns and allow for the estimation of the endogenous gene expression levels.

Role of Estrogen Response Element in the Human Prolactin Gene: Transcriptional Response and Timing.

The use of bacterial artificial chromosome (BAC) reporter constructs in molecular physiology enables the inclusion of large sections of flanking DNA, likely to contain regulatory elements and enhancers regions that contribute to the transcriptional output of a gene. Using BAC recombineering, we have manipulated a 160-kb human prolactin luciferase (hPRL-Luc) BAC construct and mutated the previously defined proximal estrogen response element (ERE) located −1189 bp relative to the transcription start site, to assess its involvement in the estrogen responsiveness of the entire hPRL locus. We found that GH3 cell lines stably expressing Luc under control of the ERE-mutated hPRL promoter (ERE-Mut) displayed a dramatically reduced transcriptional response to 17β-estradiol (E2) treatment compared with cells expressing Luc from the wild-type (WT) ERE hPRL-Luc promoter (ERE-WT). The −1189 ERE controls not only the response to E2 treatment but also the acute transcriptional response to TNFα, which was abolished in ERE-Mut cells. ERE-WT cells displayed a biphasic transcriptional response after TNFα treatment, the acute phase of which was blocked after treatment with the estrogen receptor antagonist 4-hydroxy-tamoxifen. Unexpectedly, we show the oscillatory characteristics of hPRL promoter activity in individual living cells were unaffected by disruption of this crucial response element, real-time bioluminescence imaging showed that transcription cycles were maintained, with similar cycle lengths, in ERE-WT and ERE-Mut cells. These data suggest the −1189 ERE is the dominant response element involved in the hPRL transcriptional response to both E2 and TNFα and, crucially, that cycles of hPRL promoter activity are independent of estrogen receptor binding.

Construction of a recombinant duck enteritis virus (DEV) expressing hemagglutinin of H5N1 avian influenza virus based on an infectious clone of DEV vaccine strain and evaluation of its efficacy in ducks and chickens.

BackgroundHighly pathogenic avian influenza virus (AIV) subtype H5N1 remains a threat to poultry. Duck enteritis virus (DEV)-vectored vaccines expressing AIV H5N1 hemagglutinin (HA) may be viable AIV and DEV vaccine candidates.MethodsTo facilitate the generation and further improvement of DEV-vectored HA(H5) vaccines, we first constructed an infectious clone of DEV Chinese vaccine strain C-KCE (DEVC-KCE). Then, we generated a DEV-vectored HA(H5) vaccine (DEV-H5(UL55)) based on the bacterial artificial chromosome (BAC) by inserting a synthesized HA(H5) expression cassette with a pMCMV IE promoter and a consensus HA sequence into the noncoding area between UL55 and LORF11. The immunogenicity and protective efficacy of the resulting recombinant vaccine against DEV and AIV H5N1 were evaluated in both ducks and chickens.ResultsThe successful construction of DEV BAC and DEV-H5(UL55) was verified by restriction fragment length polymorphism analysis. Recovered virus from the BAC or mutants showed similar growth kinetics to their parental viruses. The robust expression of HA in chicken embryo fibroblasts infected with the DEV-vectored vaccine was confirmed by indirect immunofluorescence and western blotting analyses. A single dose of 106 TCID50 DEV-vectored vaccine provided 100 % protection against duck viral enteritis in ducks, and the hemagglutination inhibition (HI) antibody titer of AIV H5N1 with a peak of 8.2 log2 was detected in 3-week-old layer chickens. In contrast, only very weak HI titers were observed in ducks immunized with 107 TCID50 DEV-vectored vaccine. A mortality rate of 60 % (6/10) was observed in 1-week-old specific pathogen free chickens inoculated with 106 TCID50 DEV-vectored vaccine.ConclusionsWe demonstrate the following in this study. (i) The constructed BAC is a whole genome clone of DEVC-KCE. (ii) The insertion of an HA expression cassette sequence into the noncoding area between UL55 and LORF11 of DEVC-KCE affects neither the growth kinetics of the virus nor its protection against DEV. (iii) DEV-H5(UL55) can generate a strong humoral immune response in 3-week-old chickens, despite the virulence of this virus observed in 1-week-old chickens. (iv) DEV-H5(UL55) induces a weak HI titer in ducks. An increase in the HI titers induced by DEV-vectored HA(H5) will be required prior to its wide application.

The AKR gene family and modifying sex ratios in palms through abiotic stress responsiveness.

Sex ratio (SR), the ratio of female inflorescences to total inflorescences, is one of the main yield components of oil palm (Elaeis guineensis Jacq). The SR quantitative trait locus (QTL) was recently identified on linkage (LG) 8 with a phenotype variance explained (PVE) of 11.3 %. The use of both genetic and physical mapping is one strategy for uncovering the genetic basis of the traits. Here, we report the construction of bacterial artificial chromosome (BAC) and fosmid libraries, and their use for physical mapping in oil palm. Combined, the libraries consist of more than 200,000 clones, representing 6.35 genome equivalents. Physical mapping at the SR locus was implemented by incorporating the published oil palm genome sequence and positive BAC/fosmid clones as identified by colony PCR screening. Based on the previously published sequences, the interval (about 184 kb) was comprised of 19 contigs of the known sequences (~117 kb, 64 %). After, combining the 454 pyrosequences of 15 positive clones and the previously published sequences, the known sequences were revealed to cover about 82 % of the interval (~150 kb), and were used for identifying the new markers by designing 35 gene-based and 23 simple sequence repeat (SSR)-amplified primers. As a result, a putative aldo-keto reductase gene (named EgAKR1) was revealed to be a promising candidate for sex ratio determination, via controlling female inflorescence number (11 % of PVE). This was predicted from the two newly identified polymorphic marker loci (mEgSSRsr8-21LB and mEgAKR1-9) designing from EgAKR1. The functions of AKR gene families in other plant species and our promoter analysis suggested that EgAKR1 may contribute to the sex ratio through abiotic stress responsiveness.

Differential LRRK2 expression in the cortex, striatum, and substantia nigra in transgenic and nontransgenic rodents

ABSTRACTMutations in leucine‐rich repeat kinase 2 (LRRK2) are found in a significant proportion of late‐onset Parkinson's disease (PD) patients. Elucidating the neuroanatomical localization of LRRK2 will further define LRRK2 function and the molecular basis of PD. Here, we utilize recently characterized monoclonal antibodies to evaluate LRRK2 expression in rodent brain regions relevant to PD. In both mice and rats, LRRK2 is highly expressed in the cortex and striatum, particularly in pyramidal neurons of layer V and in medium spiny neurons within striosomes. Overall, rats have a more restricted distribution of LRRK2 compared with mice. Mice, but not rats, show high levels of LRRK2 expression in the substantia nigra pars compacta. Expression of the pathogenic LRRK2‐G2019S protein from mouse bacterial artificial chromosome (BAC) constructs closely mimics endogenous LRRK2 distribution in the mouse brain. However, LRRK2‐G2019S expression derived from human BAC constructs causes LRRK2 to be expressed in additional neuron subtypes in the rat such as striatal cholinergic interneurons and the substantia nigra pars compacta. The distribution of LRRK2 from human BAC constructs more closely resembles descriptions of LRRK2 in humans and nonhuman primates. Computational analyses of DNA regulatory elements in LRRK2 show a primate‐specific promoter sequence that does not exist in lower mammalian species. These noncoding regions may be involved in directing neuronal expression patterns. Together, these studies will aid in understanding the normal function of LRRK2 in the brain and will assist in model selection for future studies. J. Comp. Neurol. 522:2465–2480, 2014. © 2014 Wiley Periodicals, Inc.

The Varicella-Zoster Virus Portal Protein Is Essential for Cleavage and Packaging of Viral DNA

The varicella-zoster virus (VZV) open reading frame 54 (ORF54) gene encodes an 87-kDa monomer that oligomerizes to form the VZV portal protein, pORF54. pORF54 was hypothesized to perform a function similar to that of a previously described herpes simplex virus 1 (HSV-1) homolog, pUL6. pUL6 and the associated viral terminase are required for processing of concatemeric viral DNA and packaging of individual viral genomes into preformed capsids. In this report, we describe two VZV bacterial artificial chromosome (BAC) constructs with ORF54 gene deletions, Δ54L (full ORF deletion) and Δ54S (partial internal deletion). The full deletion of ORF54 likely disrupted essential adjacent genes (ORF53 and ORF55) and therefore could not be complemented on an ORF54-expressing cell line (ARPE54). In contrast, Δ54S was successfully propagated in ARPE54 cells but failed to replicate in parental, noncomplementing ARPE19 cells. Transmission electron microscopy confirmed the presence of only empty VZV capsids in Δ54S-infected ARPE19 cell nuclei. Similar to the HSV-1 genome, the VZV genome is composed of a unique long region (UL) and a unique short region (US) flanked by inverted repeats. DNA from cells infected with parental VZV (VZVLUC strain) contained the predicted UL and US termini, whereas cells infected with Δ54S contained neither. This result demonstrates that Δ54S is not able to process and package viral DNA, thus making pORF54 an excellent chemotherapeutic target. In addition, the utility of BAC constructs Δ54L and Δ54S as tools for the isolation of site-directed ORF54 mutants was demonstrated by recombineering single-nucleotide changes within ORF54 that conferred resistance to VZV-specific portal protein inhibitors. Importance: Antivirals with novel mechanisms of action would provide additional therapeutic options to treat human herpesvirus infections. Proteins involved in the herpesviral DNA encapsidation process have become promising antiviral targets. Previously, we described a series of N-α-methylbenzyl-N'-aryl thiourea analogs that target the VZV portal protein (pORF54) and prevent viral replication in vitro. To better understand the mechanism of action of these compounds, it is important to define the structural and functional characteristics of the VZV portal protein. In contrast to HSV, no VZV mutants have been described for any of the seven essential DNA encapsidation genes. The VZV ORF54 deletion mutant described in this study represents the first VZV encapsidation mutant reported to date. We demonstrate that the deletion mutant can serve as a platform for the isolation of portal mutants via recombineering and provide a strategy for more in-depth studies of VZV portal structure and function.

Differential LRRK2 expression in the cortex, striatum, and substantia nigra in transgenic and nontransgenic rodents

Mutations in leucine-rich repeat kinase 2 (LRRK2) are found in a significant proportion of late-onset Parkinson's disease (PD) patients. Elucidating the neuroanatomical localization of LRRK2 will further define LRRK2 function and the molecular basis of PD. Here, we utilize recently characterized monoclonal antibodies to evaluate LRRK2 expression in rodent brain regions relevant to PD. In both mice and rats, LRRK2 is highly expressed in the cortex and striatum, particularly in pyramidal neurons of layer V and in medium spiny neurons within striosomes. Overall, rats have a more restricted distribution of LRRK2 compared with mice. Mice, but not rats, show high levels of LRRK2 expression in the substantia nigra pars compacta. Expression of the pathogenic LRRK2-G2019S protein from mouse bacterial artificial chromosome (BAC) constructs closely mimics endogenous LRRK2 distribution in the mouse brain. However, LRRK2-G2019S expression derived from human BAC constructs causes LRRK2 to be expressed in additional neuron subtypes in the rat such as striatal cholinergic interneurons and the substantia nigra pars compacta. The distribution of LRRK2 from human BAC constructs more closely resembles descriptions of LRRK2 in humans and nonhuman primates. Computational analyses of DNA regulatory elements in LRRK2 show a primate-specific promoter sequence that does not exist in lower mammalian species. These noncoding regions may be involved in directing neuronal expression patterns. Together, these studies will aid in understanding the normal function of LRRK2 in the brain and will assist in model selection for future studies.

Expression of recombinant human butyrylcholinesterase in the milk of transgenic mice

Butyrylcholinesterase (BCHE) is a natural bioscavenger that protects humans against organopho- sphate toxicity. Due to the limited yield of human BCHE (hBCHE) when purifying from human plasma, it is necessary to find an alternative method to produce this protein. One potential method is to produce transgenic livestock that make modified milk containing high concentration of hBCHE. In this study, we cloned the hBCHEgene into a human lactoferrin (hLF) bacterial artificial chromosome (BAC) construct to make a hLF- hBCHE BAC construct. Subsequently, we injected the BAC construct into pronuclei of mouse fertilized embryos and generated transgenic mice. Expression analysis showed that recombinant hBCHE (rhBCHE) was expressed effi ciently in the mammary gland of the transgenic mice and the concentration of rhBCHE in the milk of individual mice ranged from 7612 to 15928 mg$L -1 . Protein function tests showed that rhBCHE has

Plasticity in the differentiation of IL-22 producing cells (P6265)

Abstract T helper type 22 (Th22) cells are a subset of CD4+ effector T cell that primarily produces interleukin 22 (IL-22), IL-13, and TNF-alpha and have an indispensable role in host defense, barrier function, tissue repair and inflammation. However, the stability and plasticity of Th22 cells have not been elucidated. In this study we developed an IL-22-tdTomato reporter mouse to examine the plasticity of Th22 cells in vitro and in vivo. Briefly, a bacterial artificial chromosome (BAC) was modified to introduce the fluorescent reporter gene into the Il-22 locus. By homologous recombination, the synthesis of the signal peptide of Il-22 in the BAC was disrupted and the tdTomato gene with poly A was inserted immediately after the ATG start site of Il-22, replacing exon 1. Mice were generated that harbor the BAC construct. We found that in vitro generated Th22 cells still maintained their differentiation program in vitro in the early stage of development and could be reprogrammed into Th17 and Th1 cell lineages, but not Th2 cells, in the later stage. To observe Th22 plasticity in vivo, we sorted IL-22 producing cells from mouse T cell transferred colitis model and injected them back to the Rag-/- mouse. It demonstrated that Th22 cells slowly lost their IL-22 expression and converted into Th17 cells in a TGF-beta dependent manner. These results, although suggesting the flexibility of Th22 cells, also indicate active maintenance of their program in vivo.