Indexing TNF-α gene expression using a gene-targeted reporter cell line

<p>Abstract</p> <p>Background</p> <p>Current cell-based drug screening technologies utilize randomly integrated reporter genes to index transcriptional activity of an endogenous gene of interest. In this context, reporter expression is controlled by known genetic elements that may only partially capture gene regulation and by unknown features of chromatin specific to the integration site. As an alternative technology, we applied highly efficient gene-targeting with recombinant adeno-associated virus to precisely integrate a luciferase reporter gene into exon 1 of the HeLa cell tumor necrosis factor-alpha (<it>TNF-α</it>) gene. Drugs known to induce <it>TNF-α </it>expression were then used to compare the authenticity of gene-targeted and randomly integrated transcriptional reporters.</p> <p>Results</p> <p><it>TNF-α</it>-targeted reporter activity reflected endogenous <it>TNF-α </it>mRNA expression, whereas randomly integrated <it>TNF-α </it>reporter lines gave variable expression in response to transcriptional and epigenetic regulators. 5,6-Dimethylxanthenone-4-acetic acid (DMXAA), currently used in cancer clinical trials to induce <it>TNF-α </it>gene transcription, was only effective at inducing reporter expression from <it>TNF-α </it>gene-targeted cells.</p> <p>Conclusion</p> <p>We conclude that gene-targeted reporter cell lines provide predictive indexing of gene transcription for drug discovery.</p

Establishment of a Reverse Genetics System for Studying Human Bocavirus in Human Airway Epithelia

Human bocavirus 1 (HBoV1) has been identified as one of the etiological agents of wheezing in young children with acute respiratory-tract infections. In this study, we have obtained the sequence of a full-length HBoV1 genome (including both termini) using viral DNA extracted from a nasopharyngeal aspirate of an infected patient, cloned the full-length HBoV1 genome, and demonstrated DNA replication, encapsidation of the ssDNA genome, and release of the HBoV1 virions from human embryonic kidney 293 cells. The HBoV1 virions generated from this cell line-based production system exhibits a typical icosahedral structure of approximately 26 nm in diameter, and is capable of productively infecting polarized primary human airway epithelia (HAE) from the apical surface. Infected HAE showed hallmarks of lung airway-tract injury, including disruption of the tight junction barrier, loss of cilia and epithelial cell hypertrophy. Notably, polarized HAE cultured from an immortalized airway epithelial cell line, CuFi-8 (originally derived from a cystic fibrosis patient), also supported productive infection of HBoV1. Thus, we have established a reverse genetics system and generated the first cell line-based culture system for the study of HBoV1 infection, which will significantly advance the study of HBoV1 replication and pathogenesis.This work was supported by PHS R21 grant AI085236 and PHS R01 grant AI070723 from NIAID (J Qiu) and PHS R01 grant HL108902 from NHLBI (J Engelhardt)

Establishment of a Reverse Genetics System for Studying Human Bocavirus in Human Airway Epithelia

<div><p>Human bocavirus 1 (HBoV1) has been identified as one of the etiological agents of wheezing in young children with acute respiratory-tract infections. In this study, we have obtained the sequence of a full-length HBoV1 genome (including both termini) using viral DNA extracted from a nasopharyngeal aspirate of an infected patient, cloned the full-length HBoV1 genome, and demonstrated DNA replication, encapsidation of the ssDNA genome, and release of the HBoV1 virions from human embryonic kidney 293 cells. The HBoV1 virions generated from this cell line-based production system exhibits a typical icosahedral structure of approximately 26 nm in diameter, and is capable of productively infecting polarized primary human airway epithelia (HAE) from the apical surface. Infected HAE showed hallmarks of lung airway-tract injury, including disruption of the tight junction barrier, loss of cilia and epithelial cell hypertrophy. Notably, polarized HAE cultured from an immortalized airway epithelial cell line, CuFi-8 (originally derived from a cystic fibrosis patient), also supported productive infection of HBoV1. Thus, we have established a reverse genetics system and generated the first cell line-based culture system for the study of HBoV1 infection, which will significantly advance the study of HBoV1 replication and pathogenesis.</p> </div

Sequences of PCR primers designed for amplifying the terminal hairpins of HBoV1.

<p>Note: Nucleotides underlined are HBoV1 sequences or sequences complementary to the HBoV1 sequence, and nucleotides shown in bold fonts are sequences containing restriction enzyme sites and random sequences used to optimize PCR reactions and cloning.</p

IF analysis of the tight junction protein ZO-1 and the cilia marker β-tubulin IV during HBoV1 infection of primary B-HAE.

<p>Mock- and HBoV1-infected B-HAE (B33-11) cultures at the indicated days p.i. were co-stained with anti-NS1 and anti-ZO-1 (Invitrogen) antibodies (<b>A</b>), or co-stained with anti-(HBoV1) NS1 and anti-β-tubulin IV (Sigma) antibodies (<b>B</b>). Confocal images were taken at a magnification of ×40. Nuclei were stained with DAPI (blue).</p