Role of paracellular junction complexes in baculovirus-mediated gene transfer to nondividing rat hepatocytes

Bilello, John P.; Cable, Edward Earl; Myers, Roland L.; Isom, Harriet C.

doi:10.1038/sj.gt.3301937

Cited by 16 publications

(18 citation statements)

References 65 publications

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“…Longer treatment time with EGTA (40 and 60 min) detached the cell layers and thus was unsuccessful. Our data thus indicate that EGTA pretreatment only resulted in limited improvement in the gene delivery into HeLa cells and were contradictory to previous findings (Bilello et al, 2001(Bilello et al, , 2003. Also, the transduction efficiencies (f26-30%) were dramatically lower than those shown in Figure 1 (before confluency was not reached), implying the involvement of other factors.…”

Section: +contrasting

confidence: 84%

Investigation of optimal transduction conditions for baculovirus‐mediated gene delivery into mammalian cells

Hsu

Wang

et al. 2004

Biotech & Bioengineering

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Although baculovirus-mediated gene delivery into mammalian cells has been documented in a wealth of the literature, systematic investigation of the optimal transduction conditions remains unavailable. In this work, a transduction protocol using unconcentrated baculovirus is proposed for simple and efficient gene delivery into HeLa cells. We found that approximately 75-85% of the cells could be readily transduced and express the reporter protein when virus transduction occurred for 4 h at 25 degrees C using Dulbecco's phosphate-buffered saline (D-PBS) as the surrounding solution. This method contrasts with previous protocols in which transduction occurs for 1 h at 37 degrees C using growth medium (e.g., DMEM) as the surrounding solution. Investigation of the physical parameters led to the findings that: 1) baculovirus uptake by HeLa cells continued for at least 4 h in the event of high virus dosage, which led to higher gene expression; 2) the half-life of baculovirus dramatically decreased at 37 degrees C; 3) EGTA pretreatment did not apparently facilitate the gene delivery when the cells grew to multilayers; and 4) lower transduction efficiency and gene expression were obtained when DMEM was used (in comparison with D-PBS and TNM-FH), suggesting that DMEM contains certain inhibitory factors for baculovirus transduction. Our data uncovered several aspects that were not investigated before and the optimized transduction conditions allowed for gene delivery as efficient as that by the protocols commonly employed by others, but eliminated the need for virus ultracentrifugation. The protocol not only represented a simpler approach, but also considerably reduced possible virus inactivation during ultracentrifugation, thus making it easier to convert the baculovirus/mammalian cell system to a tool for eukaryotic protein production on a larger scale.

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Section: +contrasting

confidence: 84%

Investigation of optimal transduction conditions for baculovirus‐mediated gene delivery into mammalian cells

Hsu

Wang

et al. 2004

Biotech & Bioengineering

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“…Taken together, although several factors influencing baculovirus-mediated transgene expression have been examined [7,14,[39][40][41][42], herein we uncover, for the first time, that cell cycle profoundly influences the baculovirus transduction of chondrocytes. In particular, cell populations rich in quiescent cells allow for effective virus uptake, but result in inefficient nuclear transport of viral DNA and extensive DNA methylation, thus leading to poorer transgene expression.…”

Section: Discussionmentioning

confidence: 93%

Baculovirus transduction of rat articular chondrocytes: roles of cell cycle

Lee¹,

Chen²,

Shen³

et al. 2006

The Journal of Gene Medicine

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“…Cell-cell communication other than paracrine signaling by soluble mediators depends on the formation of intimate intercellular contacts through anchoring junctions (adherens junctions, desmosomes) and gap junctions [66,67].…”

Section: Communication Through Specialized Cell Junctionsmentioning

confidence: 99%

Molecular Mechanisms Underlying the Dedifferentiation Process of Isolated Hepatocytes and Their Cultures

Elaut

Henkens²,

Papeleu³

et al. 2006

CDM

260

213

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Primary hepatocytes and their cultures are a simple but versatile, well-controlled, and relatively easy to handle in vitro system that is well-accepted for investigating xenobiotic biotransformation, enzyme induction and inhibition, and (biotransformation-mediated) hepatotoxicity. In addition, hepatocyte cultures have proven to be valuable tools in the study of liver physiology, viral hepatitis, and liver regeneration and are proposed as an alternative to orthotopic liver transplantation. It has been observed, however, that a number of liver-specific functions are progressively lost with time when hepatocytes are isolated and cultivated. These phenotypic changes are primarily the result of fundamental changes in gene expression concomitant with a diminished transcription of the relevant liver-specific genes, and can be interpreted as a 'dedifferentiation' of the isolated hepatocytes. Ischemia-reperfusion stress induced during the isolation process, disruption of the normal tissue architecture, as well as an adaptation to the in vitro environment are underlying factors and will be extensively discussed. A detailed description of the regulation of the hepatocyte phenotype in vivo in the first section of this review will help to understand the effect of these factors on hepatocyte gene expression. Although different approaches, mainly mimicking the in vivo hepatocyte environment, have been succesfully used to prevent or slow down the dedifferentiation of primary hepatocytes in monolayer culture, the ideal hepatocyte-based culture model, characterized by a long-term expression of hepatocyte-specific functions comparable to the in vivo level, does not exist at the moment. Consequently, alternative strategies should focus on the isolation procedure, during which dedifferentiation is already initiated. In addition, identification of the conditions needed for the full in vitro maturation of hepatic progenitor cells to quiescent, functional hepatocyte-like cells opens promising perspectives.

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Role of paracellular junction complexes in baculovirus-mediated gene transfer to nondividing rat hepatocytes

Cited by 16 publications

References 65 publications

Investigation of optimal transduction conditions for baculovirus‐mediated gene delivery into mammalian cells

Investigation of optimal transduction conditions for baculovirus‐mediated gene delivery into mammalian cells

Baculovirus transduction of rat articular chondrocytes: roles of cell cycle

Molecular Mechanisms Underlying the Dedifferentiation Process of Isolated Hepatocytes and Their Cultures

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