A novel bacterial artificial chromosome-transgenic Podoplanin–Cre mouse targets lymphoid organ stromal cells in vivo

The steroidogenic acute regulatory protein (StAR) stimulates the regulated production of steroid hormones in the adrenal cortex and gonads by facilitating the delivery of cholesterol to the inner mitochondrial membrane. To explore key aspects of StAR function within bona fide steroidogenic cells, we used a transgenic mouse model to explore the function of StAR proteins in vivo. We first validated this transgenic bacterial artificial chromosome reconstitution system by targeting enhanced green fluorescent protein to steroidogenic cells of the adrenal cortex and gonads. Thereafter, we targeted expression of either wild-type StAR (WT-StAR) or a mutated StAR protein lacking the mitochondrial targeting signal (N47-StAR). In the context of mice homozygous for a StAR knockout allele (StAR−/−), all StAR activity derived from the StAR transgenes, allowing us to examine the function of the proteins that they encode. The WT-StAR transgene consistently restored viability and steroidogenic function to StAR−/− mice. Although the N47-StAR protein was reportedly active in transfected COS cells and mitochondrial reconstitution experiments, the N47-StAR transgene rescued viability in only 40% of StAR−/− mice. Analysis of lipid deposits in the primary steroidogenic tissues revealed a hierarchy of StAR function provided by N47-StAR: florid lipid deposits were seen in the adrenal cortex and ovarian theca region, with milder deposits in the Leydig cells. Our results confirm the ability of StAR lacking its mitochondrial targeting signal to perform some essential functions in vivo but also demonstrate important functional defects that differ from in vitro studies obtained in nonsteroidogenic cells.

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T-cell generation by lymph node resident progenitor cells

Blood ◽

10.1182/blood-2004-12-4886 ◽

2005 ◽

Vol 106 (1) ◽

pp. 193-200 ◽

Cited By ~ 35

Author(s):

Rafik Terra ◽

Isabelle Louis ◽

Richard Le Blanc ◽

Sophie Ouellet ◽

Juan Carlos Zúñiga-Pflücker ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Stromal Cells ◽

Integration Site ◽

T Cell Development ◽

Cell Development ◽

Lymphoid Organ ◽

Oncostatin M ◽

Cell Commitment

In the thymus, 2 types of Lin–Sca-1+ (lineage-negative stem cell antigen-1–positive) progenitors can generate T-lineage cells: c-Kithi interleukin-7 receptor α–negative (c-KithiIL-7Rα–) and c-KitloIL-7Rα+. While c-KithiIL-7Rα– progenitors are absent, c-KitloIL-7Rα+ progenitors are abundant in the lymph nodes (LNs). c-KitloIL-7Rα+ progenitors undergo abortive T-cell commitment in the LNs and become arrested in the G1 phase of the cell cycle because they fail both to up-regulate c-myb, c-myc, and cyclin D2 and to repress junB, p16INK4a, and p21Cip1/WAF. As a result, development of LN c-KitloIL-7Rα+ progenitors is blocked at an intermediate CD44+CD25lo development stage in vivo, and LN-derived progenitors fail to generate mature T cells when cultured with OP9-DL1 stromal cells. LN stroma can provide key signals for T-cell development including IL-7, Kit ligand, and Delta-like–1 but lacks Wnt4 and Wnt7b transcripts. LN c-KitloIL-7Rα+ progenitors are able to generate mature T cells when cultured with stromal cells producing wingless-related MMTV integration site 4 (Wnt4) or upon in vivo exposure to oncostatin M whose signaling pathway intersects with Wnt. Thus, supplying Wnt signals to c-KitloIL-7Rα+ progenitors may be sufficient to transform the LN into a primary T-lymphoid organ. These data provide unique insights into the essence of a primary T-lymphoid organ and into how a cryptic extrathymic T-cell development pathway can be amplified.

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Cloning of the Koi Herpesvirus Genome as an Infectious Bacterial Artificial Chromosome Demonstrates That Disruption of the Thymidine Kinase Locus Induces Partial Attenuation in Cyprinus carpio koi

Journal of Virology ◽

10.1128/jvi.00211-08 ◽

2008 ◽

Vol 82 (10) ◽

pp. 4955-4964 ◽

Cited By ~ 47

Author(s):

B. Costes ◽

G. Fournier ◽

B. Michel ◽

C. Delforge ◽

V. Stalin Raj ◽

...

Keyword(s):

Bacterial Artificial Chromosome ◽

Thymidine Kinase ◽

Artificial Chromosome ◽

Wild Type ◽

Bac Clone ◽

Recombinant Viruses ◽

Koi Herpesvirus ◽

Cyprinus Carpio Koi

ABSTRACT Koi herpesvirus (KHV) is the causative agent of a lethal disease in koi and common carp. In the present study, we describe the cloning of the KHV genome as a stable and infectious bacterial artificial chromosome (BAC) clone that can be used to produce KHV recombinant strains. This goal was achieved by the insertion of a loxP-flanked BAC cassette into the thymidine kinase (TK) locus. This insertion led to a BAC plasmid that was stably maintained in bacteria and was able to regenerate virions when permissive cells were transfected with the plasmid. Reconstituted virions free of the BAC cassette but carrying a disrupted TK locus (the FL BAC-excised strain) were produced by the transfection of Cre recombinase-expressing cells with the BAC. Similarly, virions with a wild-type revertant TK sequence (the FL BAC revertant strain) were produced by the cotransfection of cells with the BAC and a DNA fragment encoding the wild-type TK sequence. Reconstituted recombinant viruses were compared to the wild-type parental virus in vitro and in vivo. The FL BAC revertant strain and the FL BAC-excised strain replicated comparably to the parental FL strain. The FL BAC revertant strain induced KHV infection in koi carp that was indistinguishable from that induced by the parental strain, while the FL BAC-excised strain exhibited a partially attenuated phenotype. Finally, the usefulness of the KHV BAC for recombination studies was demonstrated by the production of an ORF16-deleted strain by using prokaryotic recombination technology. The availability of the KHV BAC is an important advance that will allow the study of viral genes involved in KHV pathogenesis, as well as the production of attenuated recombinant candidate vaccines.

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Rescue of SARS-CoV-2 from a single bacterial artificial chromosome

10.1101/2020.07.22.216358 ◽

2020 ◽

Author(s):

Chengjin Ye ◽

Kevin Chiem ◽

Jun-Gyu Park ◽

Fatai Oladunni ◽

Roy Neal Platt ◽

...

Keyword(s):

Public Health ◽

Bacterial Artificial Chromosome ◽

Reverse Genetics ◽

Cultured Cells ◽

Artificial Chromosome ◽

Natural Isolate ◽

Viral Isolate ◽

Growth Properties ◽

The City

ABSTRACTAn infectious coronavirus disease 2019 (COVID-19) emerged in the city of Wuhan (China) in December 2019, causing a pandemic that has dramatically impacted public health and socioeconomic activities worldwide. A previously unknown coronavirus, Severe Acute Respiratory Syndrome CoV-2 (SARS-CoV-2), has been identified as the causative agent of COVID-19. To date, there are no United States (US) Food and Drug Administration (FDA)-approved vaccines or therapeutics available for the prevention or treatment of SARS-CoV-2 infection and/or associated COVID-19 disease, which has triggered a large influx of scientific efforts to develop countermeasures to control SARS-CoV-2 spread. To contribute to these efforts, we have developed an infectious cDNA clone of the SARS-CoV-2 USA-WA1/2020 strain based on the use of a bacterial artificial chromosome (BAC).Recombinant (r)SARS-CoV-2 was readily rescued by transfection of the BAC into Vero E6 cells. Importantly, the BAC-derived rSARS-CoV-2 exhibited growth properties and plaque sizes in cultured cells comparable to those of the SARS-CoV-2 natural isolate. Likewise, rSARS-CoV-2 showed similar levels of replication to that of the natural isolate in nasal turbinates and lungs of infected golden Syrian hamsters. This is, to our knowledge, the first BAC based reverse genetics system for the generation of infectious rSARS-CoV-2 that displays similar features in vivo to that of a natural viral isolate. This SARS-CoV-2 BAC-based reverse genetics will facilitate studies addressing several important questions in the biology of SARS-CoV-2, as well as the identification of antivirals and development of vaccines for the treatment of SARS-CoV-2 infection and associated COVID-19 disease.

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Genotypic characterization of two bacterial artificial chromosome clones derived from a single DNA source of the very virulent gallid herpesvirus-2 strain C12/130

Journal of General Virology ◽

10.1099/vir.0.027706-0 ◽

2011 ◽

Vol 92 (7) ◽

pp. 1500-1507 ◽

Cited By ~ 17

Author(s):

Stephen J. Spatz ◽

Lorraine P. Smith ◽

Susan J. Baigent ◽

Lawrence Petherbridge ◽

Venugopal Nair

Keyword(s):

Bacterial Artificial Chromosome ◽

Artificial Chromosome ◽

Genetic Changes ◽

Mixed Genotype ◽

Bac Clones ◽

Gallid Herpesvirus 2 ◽

The Individual

The identification of specific genetic changes associated with differences in the pathogenicity of Marek's disease virus strains (GaHV-2) has been a formidable task due to the large number of mutations in mixed-genotype populations within DNA preparations. Very virulent UK isolate C12/130 induces extensive lymphoid atrophy, neurological manifestations and early mortality in young birds. We have recently reported the construction of several independent full-length bacterial artificial chromosome (BAC) clones of C12/130 capable of generating fully infectious viruses with significant differences in their pathogenicity profiles. Two of these clones (vC12/130-10 and vC12/130-15), which showed differences in virulence relative to each other and to the parental strain, had similar replication kinetics both in vitro and in vivo in spite of the fact that vC12/130-15 was attenuated. To investigate the possible reasons for this, the nucleotide sequences of both clones were determined. Sequence analysis of the two genomes identified mutations within eight genes. A single 494 bp insertion was identified within the genome of the virulent vC12/130-10 clone. Seven non-synonymous substitutions distinguished virulent vC12/130-10 from that of attenuated vC12/130-15. By sequencing regions of parental DNA that differed between the two BAC clones, we confirmed that C12/130 does contain these mutations in varying proportions. Since the individual reconstituted BAC clones were functionally attenuated in vivo and derived from a single DNA source of phenotypically very virulent C12/130, this suggests that the C12/130 virus population exists as a collection of mixed genotypes.

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Genetic Analysis of Varicella-Zoster Virus ORF0 to ORF4 by Use of a Novel Luciferase Bacterial Artificial Chromosome System

Journal of Virology ◽

10.1128/jvi.02666-06 ◽

2007 ◽

Vol 81 (17) ◽

pp. 9024-9033 ◽

Cited By ~ 60

Author(s):

Zhen Zhang ◽

Jenny Rowe ◽

Weijia Wang ◽

Marvin Sommer ◽

Ann Arvin ◽

...

Keyword(s):

Bacterial Artificial Chromosome ◽

Varicella Zoster Virus ◽

Artificial Chromosome ◽

Wild Type Virus ◽

Growth Curve Analysis ◽

Type Virus ◽

Wild Type ◽

Varicella Zoster

ABSTRACT To efficiently generate varicella-zoster virus (VZV) mutants, we inserted a bacterial artificial chromosome (BAC) vector in the pOka genome. We showed that the recombinant VZV (VZVBAC) strain was produced efficiently from the BAC DNA and behaved indistinguishably from wild-type virus. Moreover, VZV's cell-associated nature makes characterizing VZV mutant growth kinetics difficult, especially when attempts are made to monitor viral replication in vivo. To overcome this problem, we then created a VZV strain carrying the luciferase gene (VZVLuc). This virus grew like the wild-type virus, and the resulting luciferase activity could be quantified both in vitro and in vivo. Using PCR-based mutagenesis, open reading frames (ORF) 0 to 4 were individually deleted from VZVLuc genomes. The deletion mutant viruses appeared after transfection into MeWo cells, except for ORF4, which was essential. Growth curve analysis using MeWo cells and SCID-hu mice indicated that ORF1, ORF2, and ORF3 were dispensable for VZV replication both in vitro and in vivo. Interestingly, the ORF0 deletion virus showed severely retarded growth both in vitro and in vivo. The growth defects of the ORF0 and ORF4 mutants could be fully rescued by introducing wild-type copies of these genes back into their native genome loci. This work has validated and justified the use of the novel luciferase VZV BAC system to efficiently generate recombinant VZV variants and ease subsequent viral growth kinetic analysis both in vitro and in vivo.

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Systematic Excision of Vector Sequences from the BAC-Cloned Herpesvirus Genome during Virus Reconstitution

Journal of Virology ◽

10.1128/jvi.73.8.7056-7060.1999 ◽

1999 ◽

Vol 73 (8) ◽

pp. 7056-7060 ◽

Cited By ~ 233

Author(s):

Markus Wagner ◽

Stipan Jonjić ◽

Ulrich H. Koszinowski ◽

Martin Messerle

Keyword(s):

Bacterial Artificial Chromosome ◽

Artificial Chromosome ◽

Full Length ◽

Wild Type ◽

Mouse Cytomegalovirus ◽

Vector Sequences ◽

Viral Sequences ◽

Herpesvirus Genome

ABSTRACT Recently the mouse cytomegalovirus (MCMV) genome was cloned as an infectious bacterial artificial chromosome (BAC) (M. Messerle, I. Crnković, W. Hammerschmidt, H. Ziegler, and U. H. Koszinowski, Proc. Natl. Acad. Sci. USA 94:14759–14763, 1997). The virus obtained from this construct is attenuated in vivo due to deletion of viral sequences and insertion of the BAC vector. We reconstituted the full-length MCMV genome and flanked the BAC vector with identical viral sequences. This new construct represents a versatile basis for construction of MCMV mutants since virus generated from the construct loses the bacterial sequences and acquires wild-type properties.

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Construction of an infectious bacterial artificial chromosome clone of a pseudorabies virus variant: Reconstituted virus exhibited wild-type properties in vitro and in vivo

Journal of Virological Methods ◽

10.1016/j.jviromet.2018.06.004 ◽

2018 ◽

Vol 259 ◽

pp. 106-115 ◽

Cited By ~ 4

Author(s):

Tao Wang ◽

Wu Tong ◽

Chao Ye ◽

Zhiqing Yu ◽

Jing Chen ◽

...

Keyword(s):

Bacterial Artificial Chromosome ◽

Bacterial Artificial Chromosome Clone ◽

Pseudorabies Virus ◽

Artificial Chromosome ◽

Wild Type ◽

Virus Variant

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Bacterial Artificial Chromosome Mutagenesis Using Recombineering

Journal of Biomedicine and Biotechnology ◽

10.1155/2011/971296 ◽

2011 ◽

Vol 2011 ◽

pp. 1-10 ◽

Cited By ~ 21

Author(s):

Kumaran Narayanan ◽

Qingwen Chen

Keyword(s):

Gene Expression ◽

Bacterial Artificial Chromosome ◽

Restriction Enzymes ◽

Artificial Chromosome ◽

Regulatory Elements ◽

E Coli ◽

Functional Studies ◽

Large Size

Gene expression from bacterial artificial chromosome (BAC) clones has been demonstrated to facilitate physiologically relevant levels compared to viral and nonviral cDNA vectors. BACs are large enough to transfer intact genes in their native chromosomal setting together with flanking regulatory elements to provide all the signals for correct spatiotemporal gene expression. Until recently, the use of BACs for functional studies has been limited because their large size has inherently presented a major obstacle for introducing modifications using conventional genetic engineering strategies. The development ofin vivohomologous recombination strategies based on recombineering inE. colihas helped resolve this problem by enabling facile engineering of high molecular weight BAC DNA without dependence on suitably placed restriction enzymes or cloning steps. These techniques have considerably expanded the possibilities for studying functional genetics using BACsin vitroandin vivo.

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Rescue of SARS-CoV-2 from a Single Bacterial Artificial Chromosome

mBio ◽

10.1128/mbio.02168-20 ◽

2020 ◽

Vol 11 (5) ◽

Cited By ~ 2

Author(s):

Chengjin Ye ◽

Kevin Chiem ◽

Jun-Gyu Park ◽

Fatai Oladunni ◽

Roy Nelson Platt ◽

...

Keyword(s):

Bacterial Artificial Chromosome ◽

Severe Acute Respiratory Syndrome ◽

Reverse Genetics ◽

Vaccine Development ◽

Cultured Cells ◽

Artificial Chromosome ◽

Natural Isolate ◽

Growth Properties

ABSTRACT Infectious coronavirus (CoV) disease 2019 (COVID-19) emerged in the city of Wuhan (China) in December 2019, causing a pandemic that has dramatically impacted public health and socioeconomic activities worldwide. A previously unknown coronavirus, severe acute respiratory syndrome CoV-2 (SARS-CoV-2), has been identified as the causative agent of COVID-19. To date, there are no U.S. Food and Drug Administration (FDA)-approved vaccines or therapeutics available for the prevention or treatment of SARS-CoV-2 infection and/or associated COVID-19 disease, which has triggered a large influx of scientific efforts to develop countermeasures to control SARS-CoV-2 spread. To contribute to these efforts, we have developed an infectious cDNA clone of the SARS-CoV-2 USA-WA1/2020 strain based on the use of a bacterial artificial chromosome (BAC). Recombinant SARS-CoV-2 (rSARS-CoV-2) was readily rescued by transfection of the BAC into Vero E6 cells. Importantly, BAC-derived rSARS-CoV-2 exhibited growth properties and plaque sizes in cultured cells comparable to those of the natural SARS-CoV-2 isolate. Likewise, rSARS-CoV-2 showed levels of replication similar to those of the natural isolate in nasal turbinates and lungs of infected golden Syrian hamsters. This is, to our knowledge, the first BAC-based reverse genetics system for the generation of infectious rSARS-CoV-2 that displays features in vivo similar to those of a natural viral isolate. This SARS-CoV-2 BAC-based reverse genetics will facilitate studies addressing several important questions in the biology of SARS-CoV-2, as well as the identification of antivirals and development of vaccines for the treatment of SARS-CoV-2 infection and associated COVID-19 disease. IMPORTANCE The pandemic coronavirus (CoV) disease 2019 (COVID-19) caused by severe acute respiratory syndrome CoV-2 (SARS-CoV-2) is a major threat to global human health. To date, there are no approved prophylactics or therapeutics available for COVID-19. Reverse genetics is a powerful approach to understand factors involved in viral pathogenesis, antiviral screening, and vaccine development. In this study, we describe the feasibility of generating recombinant SARS-CoV-2 (rSARS-CoV-2) by transfection of a single bacterial artificial chromosome (BAC). Importantly, rSARS-CoV-2 possesses the same phenotype as the natural isolate in vitro and in vivo. This is the first description of a BAC-based reverse genetics system for SARS-CoV-2 and the first time that an rSARS-CoV-2 isolate has been shown to be phenotypically identical to a natural isolate in a validated animal model of SARS-CoV-2 infection. The BAC-based reverse genetics approach will facilitate the study of SARS-CoV-2 and the development of prophylactics and therapeutics for the treatment of COVID-19.

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