Epidermal growth factor improves lentivirus vector gene transfer into primary mouse hepatocytes

Rothe, Michael; Rittelmeyer, Ina; Iken, Marcus; Rüdrich, Urda; Schambach, Axel; Glage, Silke; Manns, Michael P.; Baum, Christopher; Bock, Michael; Ott, Michael; Modlich, Ute

doi:10.1038/gt.2011.117

Cited by 23 publications

(21 citation statements)

References 41 publications

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“…Primary mouse hepatocytes (PMHs) were isolated from BALB/cJ and/or C57BL/6J mice by a modified 2-step Liberase perfusion (21,22). Briefly, mice were anesthetized using ketamine (Albrecht) and Rompun (Bayer).…”

Section: Methodsmentioning

confidence: 99%

Control of Hepatitis C Virus Replication in Mouse Liver-Derived Cells by MAVS-Dependent Production of Type I and Type III Interferons

Anggakusuma

Frentzen

Gürlevik

et al. 2015

J Virol

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Hepatitis C virus (HCV) efficiently infects IMPORTANCEIn this study, we found that HCV NS3-4A similarly diminished both human and mouse MAVS-dependent signaling in human and mouse cells. Therefore, it is unlikely that ineffective cleavage of mouse MAVS per se precludes HCV propagation in immunocompetent mouse liver cells. Hence, approaches to reinforce HCV replication in mouse liver cells (e.g., by expression of essential human replication cofactors) should not be thwarted by the poor ability of HCV to counteract MAVS-dependent antiviral signaling. In addition, we show that mouse MAVS induces both type I and type III IFNs, which together control HCV replication. Characterization of type I or type III-dependent interferon-stimulated genes in these cells should help to identify key murine restriction factors that preclude HCV propagation in immunocompetent mouse liver cells. H epatitis C virus (HCV) infection is associated with chronic liver disease, including hepatic steatosis, fibrosis, cirrhosis, and hepatocellular carcinoma (1). Recent licensing of directly acting antivirals (DAAs) has considerably improved therapeutic options, and novel drug combinations reach cure rates of more than 90% (2). However, natural or treatment-induced virus elimination does not prevent reinfection by HCV. Moreover, many of ca. 160 million infected individuals are not diagnosed, and the vast majority of HCV patients have not been treated (3). Therefore, development of a prophylactic vaccine that efficiently prevents virus transmission is a major challenge for global control of hepatitis C. However, advances in HCV vaccine research are hampered by a lack HCV-permissive, immunocompetent animal models.HCV, a plus-strand RNA virus and member of the family Flaviviridae, has a narrow species tropism. Besides humans, only chimpanzees are naturally susceptible to chronic HCV infection. However, high costs and ethical concerns severely limit utilization of chimpanzees for research purposes. Thus, understanding of the mechanisms that limit HCV replication in other animals, for instance, mice, is an important step to ultimately develop a robust animal model for HCV. Encouragingly, recent advances have highlighted key prerequisites for HCV infection of murine liver cells. First, occludin and CD81 have been recognized to determine HCV species tropism at the level of viral cell entry (4). Importantly, this blockade can be overcome by engineering mice to express human occludin and CD81 (5) or by adaptation of HCV to usage of mouse CD81 (6). Second, HCV replication in murine embryonic fibroblasts (7,8), in mouse liver-derived cells (9), and

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Section: Methodsmentioning

confidence: 99%

Control of Hepatitis C Virus Replication in Mouse Liver-Derived Cells by MAVS-Dependent Production of Type I and Type III Interferons

Anggakusuma

Frentzen

Gürlevik

et al. 2015

J Virol

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“…Vector-derived copy numbers were determined by real-time PCR as described previously (18,19). A total of 100 ng/2 ml genomic DNA prepared from the above step were added to the 13-ml RQ-PCR mix containing 7.5 ml SYBRTaq mix with 1 ml primer mix for woodchuck hepatitis virus posttranscriptional regulatory element, 59-GAGGAGTTGTGGCCCGTTGT-39 (forward), and 59-TGACA-GGTGGTGGCAATGCC-39 (reverse); or polypyrimidine tract binding protein 2, 59-TCTCCATTCCCTATGTTCATGC-39 (forward), and 59-GTTCCCGCAGAATGGTGAGGTG-39 (reverse), adjusting the volume to 13 ml with PCR-grade nuclease-free water.…”

Section: Real-time Pcr For Analyses Of Lentiviral Vector Copies In Timentioning

confidence: 99%

Dendritic Cell–Mediated Immune Humanization of Mice: Implications for Allogeneic and Xenogeneic Stem Cell Transplantation

Salguero

Daenthanasanmak

Münz

et al. 2014

The Journal of Immunology

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De novo regeneration of immunity is a major problem after allogeneic hematopoietic stem cell transplantation (HCT). HCT modeling in severely compromised immune-deficient animals transplanted with human stem cells is currently limited because of incomplete maturation of lymphocytes and scarce adaptive responses. Dendritic cells (DC) are pivotal for the organization of lymph nodes and activation of naive T and B cells. Human DC function after HCT could be augmented with adoptively transferred donor-derived DC. In this study, we demonstrate that adoptive transfer of long-lived human DC coexpressing high levels of human IFN-α, human GM-CSF, and a clinically relevant Ag (CMV pp65 protein) promoted human lymphatic remodeling in immune-deficient NOD.Rag1−/−.IL-2rγ−/− mice transplanted with human CD34+ cells. After immunization, draining lymph nodes became replenished with terminally differentiated human follicular Th cells, plasma B cells, and memory helper and cytotoxic T cells. Human Igs against pp65 were detectable in plasma, demonstrating IgG class-switch recombination. Human T cells recovered from mice showed functional reactivity against pp65. Adoptive immunotherapy with engineered DC provides a novel strategy for de novo immune reconstitution after human HCT and a practical and effective tool for studying human lymphatic regeneration in vivo in immune deficient xenograft hosts.

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“…3B) but were actually present at low levels; the 10 other samples showed a low correlation (r = 0.138, p = 0.6687) with qPCR data. Furthermore, this experiment showed that in a set of insertions in which we expected no or little clonal expansion because these hepatocytes failed to divide in vitro (Rothe et al, 2012), we did find clones with associated high numbers of reads (Fig. 3A), which would have led us to the erroneous conclusion that expansion of several individual clones did occur.…”

Section: Lm-pcr: 454 Pyrosequencing Sensitivity Compared With Site-spmentioning

confidence: 90%

Evaluating a Ligation-Mediated PCR and Pyrosequencing Method for the Detection of Clonal Contribution in Polyclonal Retrovirally Transduced Samples

Brugman

Suerth

Rothe

et al. 2013

Human Gene Therapy Methods

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Retroviral gene transfer has proven therapeutic potential in clinical gene therapy trials but may also cause abnormal cell growth via perturbation of gene expression in the locus surrounding the insertion site. By establishing clonal marks, retroviral insertions are also used to describe the regenerative potential of individual cells. Deep sequencing approaches have become the method of choice to study insertion profiles in preclinical models and clinical trials. We used a protocol combining ligation-mediated polymerase chain reaction (LM-PCR) and pyrosequencing for insertion profiling and quantification in cells of various tissues transduced with various retroviral vectors. The presented method allows simultaneous analysis of a multitude of DNA-barcoded samples per pyrosequencing run, thereby allowing cost-effective insertion screening in studies with multiple samples. In addition, we investigated whether the number of pyrosequencing reads can be used to quantify clonal abundance. By comparing pyrosequencing reads against site-specific quantitative PCR and by performing spike-in experiments, we show that considerable variation exists in the quantification of insertion sites even when present in the same clone. Our results suggest that the protocol used here and similar approaches might misinterpret abundance clones defined by insertion sites, unless careful calibration measures are taken. The crucial variables causing this variation need to be defined and methodological improvements are required to establish pyrosequencing reads as a quantification measure in polyclonal situations.

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Epidermal growth factor improves lentivirus vector gene transfer into primary mouse hepatocytes

Cited by 23 publications

References 41 publications

Control of Hepatitis C Virus Replication in Mouse Liver-Derived Cells by MAVS-Dependent Production of Type I and Type III Interferons

Control of Hepatitis C Virus Replication in Mouse Liver-Derived Cells by MAVS-Dependent Production of Type I and Type III Interferons

Dendritic Cell–Mediated Immune Humanization of Mice: Implications for Allogeneic and Xenogeneic Stem Cell Transplantation

Evaluating a Ligation-Mediated PCR and Pyrosequencing Method for the Detection of Clonal Contribution in Polyclonal Retrovirally Transduced Samples

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