Enhancement of Gene Expression by Somatic Hybridization with Primary Cells: High-Level Synthesis of the Hepatitis B Surface Antigen in Monkey Vero Cells by Fusion with Primary Hepatocytes

Chenciner, Nicole; Delpeyroux, Françis; Israël, Nicole; Lambert, Mireille; Lim, Annick; Streeck, Rolf E.; Houssais, Jean‐François

doi:10.1038/nbt0990-858

Cited by 4 publications

(2 citation statements)

References 19 publications

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“…The plasmid pHBV⌬core harbors the same ayw genome and allows HBsAg expression, but is unable to replicate because of a deletion in the core gene (25).…”

mentioning

confidence: 99%

Extensive editing of both hepatitis B virus DNA strands by APOBEC3 cytidine deaminases in vitro and in vivo

Suspène

Guétard

Mathieu

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

280

266

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Because the replication of hepatitis B virus (HBV) proceeds via an obligatory reverse transcription step in the viral capsid, cDNA is potentially vulnerable to editing by cytidine deaminases of the APOBEC3 family. To date only two edited HBV genomes, referred to as G 3 A hypermutants, have been described in vivo. Recent work suggested that HBV replication was indeed restricted by APOBEC3G but by a mechanism other than editing. The issue of restriction has been explored by using a sensitive PCR method allowing differential amplification of AT-rich DNA. G 3 A hypermutated HBV genomes were recovered from transfection experiments involving APOBEC3B, -3C, -3F, and -3G indicating that all four enzymes were able to extensively deaminate cytidine residues in minus-strand DNA. Unexpectedly, three of the four enzymes (APOBEC3B, -3F, and -3G) deaminated HBV plus-strand DNA as well. From the serum of two of four patients with high viremia, G 3 A hypermutated genomes were recovered at a frequency of Ϸ10 ؊4 , indicating that they are, albeit relatively rare, part of the natural cycle of HBV infection. These findings suggest that human APOBEC3 enzymes can impact HBV replication via cytidine deamination.hypermutation ͉ DNA editing G 3 A hypermutated retroviral genomes result from the editing of nascent DNA by APOBEC3 cytidine deaminases (1-7). This was originally demonstrated for HIV-1 and the APOBEC3G member of a cluster of seven genes (3A-3H) on human chromosome 22 (8, 9). Editing occurs on the background of a ⌬vif genome (1-5, 10). The HIV Vif protein prevents packaging of either APOBEC3F or -3G resulting in their ubiquitination and degradation by the proteasomal pathway (11)(12)(13)(14). Editing of cytidine residues in neo-synthesized minusstrand cDNA results in the formation of multiple uracil residues that are read as T during plus-strand synthesis. Albeit referred to as G 3 A hypermutants by reference to the viral plus strand, mechanistically the action is occurring on the minus strand and independently of reverse transcriptase (7). Up to 60% of G residues in a lentiviral genome can be substituted resulting in the total loss of information (15-17). As such, APOBEC3F and -3G constitute a powerful restriction mechanism to reverse transcription. Retroviruses have either to avoid replication in cells expressing APOBEC3 molecules or else evolve a mechanism that neutralizes their effect. The vif gene of human and primate lentiviruses, as well as their homologues in most of the other lentiviruses, reflect the latter solution.Hepatitis B viruses (HBVs) replicate via an obligate reverse transcription step occurring in a capsid structure close to the endoplasmic reticulum. A pair of G 3 A hypermutated genomes were identified in the blood of a chronically infected patient yet have remained unique despite a burgeoning database (18). Recent reports demonstrated that HBV replication could be strongly restricted by APOBEC3G and -3F in an experimental setting (20,21). Restriction was highlighted by a strong reduction in the proportion o...

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“…The plasmid pHBV⌬core harbors the same ayw genome and allows HBsAg expression, but is unable to replicate because of a deletion in the core gene (25).…”

mentioning

confidence: 99%

Extensive editing of both hepatitis B virus DNA strands by APOBEC3 cytidine deaminases in vitro and in vivo

Suspène

Guétard

Mathieu

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

280

266

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“…The most common use is in the establishment of hybridoma cells for the production of monoclonal antibodies. Cell fusion as a method of enhancing protein production has been described by Chenciner et al, 1990. They produced classical hybrids between a liver cell and a transfected Vero cell.…”

Section: Introductionmentioning

confidence: 99%

Untitled

Dyring¹

1997

Cytotechnology

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A recombinant Chinese hamster ovary (CHO) cell clone, S1, stably expressing human insulin-like growth factor binding protein-1 (hIGFBP-1), was treated with polyethylene glycol (PEG), resulting in cell fusion, in order to further enhance the protein expression by increasing the gene copy number and/or the amount of organelles important to the protein expression/-secretion. Both the fused cell line, Peg1, and its mother cell line, S1, were adapted to serum-free growth in suspension and were characterised with respect to growth and productivity. Peg1 was easier to adapt to the serum-free suspension conditions and had a higher viability during the adaptation period than S1. Furthermore, Peg1 showed a stable productivity of hIGFBP-1 that was twice as high as that for S1 under both adherent and suspension conditions. A considerable difference in the specific productivity (up to 3-4 times) was noticed during the growth phase. PEG fusion experiments have earlier been studied in our laboratory with CHO cells producing recombinant factor VIII and our results correlates very well with the results obtained with the factor VIII producing cells. Surprisingly, it was possible to obtain high producing recombinant cell lines, which were stable for more than 4 months.

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Enhancement of γ -Carboxylation of Recombinant Activated Protein C by Cell Fusion

Takazawa

Wakabayashi

Miyagi

et al. 1994

Animal Cell Technology: Basic &Amp; Applied Aspects

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Enhancement of Gene Expression by Somatic Hybridization with Primary Cells: High-Level Synthesis of the Hepatitis B Surface Antigen in Monkey Vero Cells by Fusion with Primary Hepatocytes

Cited by 4 publications

References 19 publications

Extensive editing of both hepatitis B virus DNA strands by APOBEC3 cytidine deaminases in vitro and in vivo

Extensive editing of both hepatitis B virus DNA strands by APOBEC3 cytidine deaminases in vitro and in vivo

Untitled

Enhancement of γ -Carboxylation of Recombinant Activated Protein C by Cell Fusion

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