Abstract
Isolation of hepatocytes and their culture in vitro represent important avenues to explore the function of such cells. However, these studies are often difficult to perform because of the inability of hepatocytes to proliferate in vitro. Immortalization of isolated hepatocytes is thus an important step toward continuous in vitro culture. For cellular immortalization, integration of relevant genes into the host chromosomes is a prerequisite. Transposons, which are mobile genetic elements, are known to facilitate integration of genes of interest (GOI) into chromosomes in vitro and in vivo. Here, we proposed that a combination of transposon- and liver-directed introduction of nucleic acids may confer acquisition of unlimited cellular proliferative potential on hepatocytes, enabling the possible isolation of immortalized hepatocyte cell lines, which has often failed using more traditional immortalization methods.
Highlights
It is well-known that primary cells can only undergo a limited number of cell divisions in culture, the number varies according to species, cell type, and culture conditions [1]
This replicative senescence can be overcome by overexpression of viral genes such as simian virus 40 large T antigen (SV40T) [4] or telomerase reverse transcriptase protein (TERT), the latter being responsible for elongation of telomeres [5, 6], through in vitro transfection of primary cells
We found that expression of a genes of interest (GOI) continued for at least 1.5 months after intrapancreatic parenchymal injection for gene transfer (IPPIGT)
Summary
It is well-known that primary cells can only undergo a limited number of cell divisions in culture, the number varies according to species, cell type, and culture conditions [1]. In earlier stages of attempts to acquire immortalized hepatocytes, many researchers employed viral infection approaches involving primary cultured hepatic cells obtained soon after isolation from liver tissue after perfusion with collagenase (Figure 1(a)). These viruses include adenovirus and SV40 virus containing oncogenic factors such as E1A/E1B (adenovirus) and transforming genes (SV40) [15,16,17]. Many of the immortalized hepatocyte lines established by the above-mentioned technologies appear to lose hepatocyte-specific functions, as exemplified by reduced production of albumin, urea, and cytochromes, compared to the living liver Almost all of these cells lose infectivity by HBV except the HuS-E/2 human hepatocyte cell line [52]. One reason for this failure appears to be due to in vitro immortalization of in vitro cultured hepatocytes
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