Transfection of hepatic genes into adult rat hepatocytes in primary culture and their tissue-specific expression

We describe in this paper a method for studying transient gene expression in a primary culture of adult rat hepatocytes. After isolation by collagenase perfusion, hepatocytes in a monolayer were transfected with foreign DNA by the calcium phosphate precipitation technique during the first 24 hours after plating. When they were transfected with a plasmid containing the gene for chloramphenicol acetyltransferase driven by the early promoter of simian virus 40, hepatocytes reproducibly expressed high levels of chloramphenicol acetyltransferase (CAT); this transient expression was much higher than that obtained with the rat hepatoma cell line H4II. Different medium conditions have been tested; an optimal level of CAT activity can be obtained using a serum-free, hormonally defined medium. Using these techniques, we have investigated the expression of liver-specific genes transferred into hepatocytes. We show that the L-pyruvate kinase promoter is active in these hepatocytes while it is silent in fibroblasts. Moreover, the use of serum-free medium may allow investigation of the role of hormones and nutrients in cells which respond normally to these effectors.     

Transient expression of the CD2 cell surface antigen as a sortable marker to monitor high frequency transfection of human primary B cells.

We have used the T cell surface molecule CD2 gene, expressed from the human cytomegalovirus promoter as a reporter to optimize a transfection system for human primary B cells. The CD2-encoding DNA was transfected into cells by electroporation and transient expression was monitored by flow cytometric analysis. By using our optimal electroporation conditions on activated primary B cells, more than 30% of the resulting viable cells expressed CD2 on the cell surface. Moreover, unactivated primary B cells could also be transfected using this system but subsequent expression of CD2 required cellular activation. Magnetic beads or plastic culture bottles coated with anti-CD2 antibodies have been used to selectively purify transfected cells. The high transfection efficiency combined with the ability to specifically purify transfected cells may allow future studies on specific genes transiently expressed in human primary B cells.     

Electroporation-mediated gene transfer into hepatocytes: preservation of a growth hormone response.

An electroporation procedure is described which allows the introduction of foreign genes into freshly isolated rat hepatocytes while preserving their growth hormone responsiveness. A single-pulse procedure performed at low voltage (150-200 V) but with high capacitance (960 microF), conditions which caused minimal cell damage and increased hepatocyte survival in culture (greater than 80%), was found to be optimal for both the basal and the hormone-stimulated expression of transfected genes. Transfection of the cells suspended in a phosphate buffer at high concentrations (20-25 x 10(6)/ml) with large amounts of plasmid (30 micrograms/assay) gave the best results. Raising the temperature up to 25 or 37 degrees C (instead of 4 degrees C) decreased about twofold basal CAT expression but appeared to increase the magnitude (i.e., fold induction) of hormonal effects. Expression of the reporter gene driven by either a viral or a liver gene promoter reached a maximum after 24 h, a situation especially favorable when studying liver-specific gene expression known to decay rapidly in cultured hepatocytes. This procedure was successfully applied to the study of a growth hormone-dependent serine protease inhibitor gene promoter.     

Transformation of differentiated rat hepatocytes with adenovirus and adenovirus DNA

Primary cultures of hepatocytes isolated by collagenase perfusion of adult rats were transformed by infection with adenovirus type 5 or transfection with adenovirus DNA. Total virion DNA or recombinant plasmid DNA containing the adenovirus E1A and E1B genes transformed hepatocytes at comparable frequencies. No foci of replicating hepatocytes were detected after transfection with a plasmid containing the E1A gene alone. The frequency of transformation by the adenovirus E1A and E1B genes was dependent on the composition of the culture medium. Transformation occurred at a low frequency when the transfected hepatocytes were maintained in a chemically defined medium (CDM), but the frequency was enhanced 8- to 10-fold when the cells were maintained in (i) serum-supplemented medium or (ii) CDM supplemented with epidermal growth factor. Cell lines derived from the adenovirus-transformed colonies of hepatocytes expressed adenovirus E1A and E1B RNAs. When hepatocytes were maintained in CDM supplemented with dimethyl sulfoxide and transfected with plasmids containing the E1A and E1B genes, it was possible to derive cell lines that retained the ability to express several liver-specific genes, including albumin, transferrin, hemopexin, and the third component of complement. The amount of albumin secreted per cell varied from 1 to 5 pg per cell per 24 h, and in one cell line it was below detectable levels by passage 9. Adenovirus-transformed hepatocytes were not tumorigenic when inoculated subcutaneously into neonatal syngeneic rats. We conclude that the adenovirus E1A and E1B genes are capable of transforming adult rat hepatocytes, a differentiated epithelial cell type.     

An epoch-making application of discharge plasma phenomenon to gene-transfer

We discovered an epoch-making gene transfer method utilizing discharge plasma. Although an electroporation method is commonly used in present gene transfer experiments, it cannot transfer genes into primary cells sufficiently. The atmospheric pressure discharge plasma employed in this study was originally used for surface treatment of non-biological materials. We hypothesized that it could provide a suitable effect on the surface of target cells and applied it to gene transfer into various types of cells. The plasma technology succeeded in the efficient transfer of green fluorescence protein (GFP) plasmid into post-mitotic neuronal cells obtained from cerebral cortices of rats, into which an electroporation with conventional equipment cannot transfer genes sufficiently, as the cells were attached. After the transfection of rat pheochromocytoma PC12 cells with the GFP gene by plasma treatment, the cells retained their function, that is, nerve growth factor-induced differentiation. Furthermore, gene transfer with the plasma technology was also applicable to other types of cell lines such as HeLa cells and Chinese hamster lung (CHL) cells as adherent cell lines, and Jurkat cells as a suspended cell line, and another type of primary cell, human umbilical vein endothelial cells (HUVEC). In conclusion, the plasma method is an epoch-making gene transfer technology which efficiently transfers genes into primary cells into which electroporation cannot transfer genes. Moreover, the method is able to universally transfer genes into various types of cells as the function of the cells was maintained.     

Electroporation of primary neural cultures: a simple method for directed gene transfer in vitro

Gene transfer into cells of the nervous system is an important method to analyze tissue-specific gene functions. Although highest transfection efficiencies are generally obtained by viral gene transfer, non-viral methods are attractive because they are less labor intensive and more suitable for high throughput screening approaches. Here we describe an approach for electroporation-based gene transfer into primary neural cells isolated from dissociated murine cerebella. Using GFP as reporter molecule, we show that electroporation allows for efficient gene transfer into embryonic and postnatal neural cells under highly controlled experimental conditions. Furthermore we show that adaptation of electroporation parameters allowed for the preferential transfection of subsets of neural cells within the mixed primary culture. Using electroporation settings of high voltage and low capacitance (500 V/50 microF) we achieved a transfection efficiency of about 10% of small neural cells which were identified as granule cells by the expression of the granule cell-specific marker NeuN. At electroporation settings of 220 V/975 microF, large and stellate-shaped cells that comprised about 10% of the GFAP-positive population of astrocytes were preferentially transfected. We conclude that electroporation of primary neural cells can be used to target gene transfer to subsets of neural cells.     

Immortalization of rat kidney glomerular mesangial cell and its coculture with glomerular epithelial cell

Mesangial cell has several key roles in the control of glomerular function: it participates in the regulation of glomerular filtration rate, macromolecular clearance, and as both a source and target of numerous hormones and autocrines. Many of these insights into mesangial cell function have been obtained by studying mesangial cells in culture. However, no suitable cell lines have been established yet. We here reported the immortalization of rat kidney glomerular mesangial cell by transfection of E6 and E7 genes of human papillomavirus type 16 (HPV-16) via electroporation and lipofection. The results showed that only electroporation could transfect the genes to mesangial cells and the transfected cells maintained the viability for longer than 6 months. Fluorescence microscopic observation showed that cellular contractility and phagocytosis, which are the two main phenotypes of mesangial cells, are well maintained after transfection. The coculture of transfected mesangial cells with rat glomerular epithelial cells showed that the growth of mesangial cells was suppressed by epithelial cell, but the growth of epithelial cells was enhanced by mesangial cells. Moreover, an enhancing effect on the phagocytosis of mesangial cell was also observed in coculture. Such results may imply that the glomerular cell-cell interaction plays an important role in the regulation of cell proliferation and differentiation.     

Down-regulation of survival signaling through MAPK and Akt in occludin-deficient mouse hepatocytes in vitro

The tight junction (TJ) regulates epithelial cell polarity and barrier including permeability of the paracellular pathway. Occludin was the first integral membrane protein to be discovered, but it is not indispensable for the formation of TJ strands. The physiological function of occludin is still unclear, although occludin-deficient mice show very complex abnormalities in various organs without overt dysfunction of the TJ. To investigate the role of occludin in TJ expression and apoptosis regulated by survival signal transduction pathways such as MAPK and Akt, we performed primary culture of hepatocytes and established hepatic cell lines from occludin-deficient mice. In primary cultures of occludin-deficient mouse hepatocytes, claudin-2 expression and apoptosis were induced by down-regulation of the activation of MAPK and Akt. In the hepatic cell lines derived from occludin-deficient mice, claudin-2 expression and serum-free induced apoptosis were also increased by down-regulation of the activation of MAPK and Akt. Furthermore, in the hepatic cell lines transiently transfected with mouse and rat occludin genes, induction of claudin-2 expression and the apoptosis were inhibited with increases in activation of MAPK and Akt. These findings show that occludin plays a crucial role in claudin-2-dependent TJ function and the apoptosis involving MAPK and Akt signaling pathways in hepatocytes.     

Gene transfer into established and primary fibroblast cell lines: comparison of transfection methods and promoters.

The stable transfection of immortalized Rat-1 and rat skin primary fibroblast cell lines by calcium phosphate precipitation, lipofection and electroporation methods have been examined. The lipofection method was found to be better than the other methods in terms of higher transfection efficiency and convenient use. Expression of beta-galactosidase from two different viral promoters showed that the level of transgene expression depends on the promoter strength in a particular cell type. The results presented here show that the transgene expression is extremely variable among different colonies generated from individually transfected cells. Therefore, it is necessary to examine individual colonies of cells for the production of reporter gene to obtain cell lines expressing high amounts of gene products.     

Cationic lipid-mediated transfection of liver cells in primary culture.

We describe transfection of DNA into parenchymal and individual non-parenchymal cell populations from adult rat liver in early primary culture, using cationic lipid as the carrier. All cell populations were transfectable, although lipid requirements varied by cell type and, for hepatocytes, with the age of the culture. For hepatocytes in early primary culture (2-10 hours after plating), pure DOTMA (N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride) was strikingly more effective than commercial formulations (Lipofectin or TransfectACE) containing components in addition to, or other than DOTMA. For hepatocytes fully adapted to culture (approximately 48 hours after plating), pure DOTMA and Lipofectin were similarly effective. Under optimal conditions, about 10% of hepatocytes expressed the transfected reporter gene. CAT expression in hepatocytes doubled from 48 hours to 7 days after transfection. The effect of culture substratum on transfection efficiency also was examined. The presence of basement membrane-like matrix (EHS gel) reduced uptake of the DNA-lipid complex. However, cells in early culture that were transfected on collagen and then replated on EHS gel, displayed significantly greater reporter gene activity than did cells maintained throughout on collagen. In contrast to hepatocytes, non-parenchymal cells (lipocytes, Kupffer cells and endothelial cells, respectively) were transfected most efficiently by Lipofectin; DOTMA alone was inactive. The methods described will facilitate studies of gene regulation in individual liver cell populations.     

Maintenance of liver function in long term culture of hepatocytes following In Vitro or In Vivo Ha-ras EJ transfection

Collagenase isolated rat hepatocytes were transfected with liposome encapsulated pEJ (LE-pEJ), a plasmid carrying the human cellular activated Ha-ras^EJ oncogene. A proliferative cell line was cloned from these cells transfected in vitro. It secreted per day 0.87 µg albumin and 0.32 µg transferrin per 10⁶ cells, and 11.06 nmol free and conjugated bile acids (BA) per mg protein. Also, it metabolized 2-acetylaminoflourene (2-AFAF) into N- and ring-hydroxylated metabolites and 2-aminofluorene at rates of 1.50, 9.73, and 1.98 nmol/mg cell protein/24 hr, respectively. Rats were i.v. injected with both LE-pEJ and LE-p17hGHnneo carrying the hGH cDNA gene, and secreted hGH in the plasma which induced the synthesis of anti-hGH antibodies. A cell line was cloned from cultures of primary hepatocytes isolated from the liver of transfected rats. After 2 to 3 months in culture, this cell line secreted per day 18.9 µg albumin and 11.0 µg transferrin per 10⁶ cells, 38.75 nmol total BA per mg cell protein, and up to 31 ng hGHper 10⁶ cells without cloning hGH recombinant cells. A 24 hr control culture of primary hepatocytes isolated from non transfected rats secreted 25.5 µg albumin and 11.7 µg transferrin per 10⁶ cells, and produced 21.64 nmol total BA and 2.13 nmol N-OH-2-AFAF per mg cell protien. Hence, Ha-ras ^EJ transfection of either hepatocytes in vitro or liver cells in vivo, initiated cell cycles leading to presumptive proliferating hepatocytes which express liver function.Abbreviations BWE basal Williams' medium E - FBS fetal bovine serum - F10 or F12 basal Ham's F10 or F12 medium - Ha-ras ^EJ EJ allele of the human cellular ras oncogen of Harvey - hGH human growth hormone - hsp heat shock protein gene - LE-p liposome encapsulated plasmid - N-OH-2-AFAF N-hydroxy-2-acetylaminofluorene - RLECC rat liver epithelial cell - SF serum-free - SS serum-supplemented - UGG serum substitute UGltroser G® - 1-OH-, 3-OH-2-AFAFF 1-hydroxy-, 3-hydroxy-2-acetylaminofluorene - 2-AFAF 2-acetylaminofluorene - 2-AFF 2-aminofluorene     

原代大鼠海马神经元的高效转染

用原代大鼠海马神经元为模型,对新型电转染方法Nucleofector^TM与脂质体DOTAP和Lipofectaimine^TM的转染效率和转染前后细胞存活率进行比较研究,探讨Nucleofector^TM的高效性与可靠性。从E18胎鼠海马中取出神经元进行体外培养,并用神经微丝(NF)抗体进行免疫细胞化学染色鉴定细胞类型。分别用DOTAP,Lipofectamine^TM and Nucleofector^TM包裹pCMV-eGFP质粒转染原代大鼠海马神经元。神经元的存活率用流式细胞仪检测。实验结果表明：DOTAP和Lipofectamine^TM的基因转染效率仅为1．55％和2．45％,而Nucleofector^TM的转染效率则超过20％;细胞转染前后的存活率在DOTAP组分别为98．37％和88．35％,Lipofectamine^TM组分别为98．37％和90．11％,而在Nucleofector^TM组中分别为98．37％和51．82％。上述实验数据表明：Nucleofector^TM转染技术能高效并安全地转染原代大鼠海马神经元,但死亡率较高。     

Analysis of glutathione transferase P gene regulation with liver cells in primary culture.

Glutathione transferase P (GST-P) gene is specifically and highly activated during rat chemical hepatocarcinogenesis. We have previously cloned the GST-P gene and have identified putative regulatory regions. To further explore regulatory mechanisms, deletion constructs of the GST-P gene fused to the chloramphenicol acetyltransferase (CAT) structural gene were introduced into primary cultured rat hepatocytes by electroporation, and their activity was determined. The expression of the GST-P-CAT fusion gene is quite low in these cells as compared to that in both a rat fibroblast cell line, 3Y1 cells, and a rat hepatoma cell line, dRLh84. The presence of the strong enhancer GPEI did not elicit any enhancing activity at its original position, or when it was located 3' of the CAT gene, although this element does enhance CAT activity significantly when located adjacent to the promoter. Cotransfection of neither c-jun nor c-fos expression vector, nor both vectors, could enhance the CAT activity, even though GPEI consists of two phorbol ester response element-like sites. Furthermore, the expression of jun family gene was not correlated with GST-P gene expression either in primary cultured hepatocytes or in hepatoma cell lines.     

Electroporation optimization to deliver plasmid DNA into dental follicle cells

Electroporation is a simple and versatile approach for DNA transfer but needs to be optimized for specific cells. We conducted square wave electroporation experiments for rat dental follicle cells under various conditions. These experiments indicated that the optimal electroporation electric field strength was 375 V/cm, and that plasmid concentrations greater than 0.18 μg/μL were required to achieve high transfection efficiency. BSA or fetal bovine serum in the pulsing buffer significantly improved cell survival and increased the number of transfected cells. The optimal pulsing duration was in the range of 45–120 ms at 375 V/cm. This electroporation protocol can be used to deliver DNA into dental follicle cells to study the roles of candidate genes in regulating tooth eruption. This is the first report showing the transfection of dental follicle cells using electroporation. The parameters determined in this study are likely to be applied to transfection of other fibroblast cells.     

Depot-dependent effects of adipose tissue explants on co-cultured hepatocytes

We have developed an in vitro hepatocyte-adipose tissue explant (ATE) co-culture model enabling examination of the effect of visceral and subcutaneous adipose tissues on primary rat hepatocytes. Initial analyses of inflammatory marker genes were performed in fractionated epididymal or inguinal adipose tissues. Expressions of inflammation related genes (IL-6, TNF-α, COX-2) were higher in the inguinal than the epididymal ATE. Similarly, expressions of marker genes of macrophage and monocyte (MPEG-1, CD68, F4/80, CD64) were higher in the stromal vascular fraction (SVF) isolated from inguinal ATE than that from epididymal ATE. However, expressions of lipolysis related genes (ATGL, HSL, perilipin-1) were higher in the epididymal adipocytes than inguinal adipocytes. Moreover, secretion of IL-6 and PGE(2) was higher from inguinal ATEs than from epididymal ATEs. There was a trend that the total levels of IL-6, TNF-α and PGE(2) in the media from inguinal ATEs co-cultured with primary rat hepatocytes were higher than that in the media from epididymal ATEs co-cultured with hepatocytes, although the significant difference was only seen in PGE(2). Lipolysis, measured as glycerol release, was similar in the ATEs isolated from inguinal and epididymal adipose tissues when cultured alone, but the glycerol release was higher in the ATEs isolated from epididymal than from inguinal adipose tissue when co-cultured with hepatocytes. Compared to epididymal ATEs, the ATEs from inguinal adipose tissue elicited a stronger cytotoxic response and higher level of insulin resistance in the co-cultured hepatocytes. In conclusion, our results reveal depot-dependent effects of ATEs on co-cultured primary hepatocytes, which in part may be related to a more pronounced infiltration of stromal vascular cells (SVCs), particularly macrophages, in inguinal adipose tissue resulting in stronger responses in terms of hepatotoxicity and insulin-resistance.     

Characterization of the 5' flanking region of rat glucokinase gene

The rat glucokinase (GK) gene containing the first exon was isolated and its 5' flanking region was characterized by the bacterial chloramphenicol acetyltransferase (CAT) assay. A transient expression assay with a series of 5' deletion constructs (-5.5 k to -48) of GK-CAT fusion genes indicated that the 5' flanking sequence up to nucleotide -87 was sufficient for promoter activity in adult rat hepatocytes, but its activity was much weaker than that of the SV40 enhancer/promoter. Similar promoter activity was also detected in dRLh-84 hepatoma cells, which do not express glucokinase. Insulin treatment caused no change in the CAT activity of hepatocytes transfected with the fusion genes. These results suggest that the 5' flanking region of the glucokinase gene up to -5.5 k does not contain enhancer elements responsible for tissue-specific expression or insulin regulation.     

Multiplicative mononuclear small hepatocytes in adult rat liver: their isolation as a homogeneous population and localization to periportal zone

Adult rat liver contains a minor population of hepatocytes called small hepatocytes (SHs) that are smaller in size and show a higher replicative potential than conventional parenchymal hepatocytes (PHs). However, SHs have been hitherto characterized using a "SH-fraction" that was contaminated with PHs. In the present study, we isolated a PH-free SH-fraction from the adult rat liver using fluorescence-activated cell sorter combined with centrifugal elutriation and characterized the hepatocytes in the fraction. These hepatocytes were designated R3Hs in this study. R3Hs were mononuclear and of lower ploidy. They expressed at high levels genes of Cdc2, connexin 26, hydroxysteroid sulfotransferase, pancreatic secretory trypsin inhibitor, and prostaglandin E2 receptor EP3 subtype. We conclude that SHs dominate the periportal zone in the adult liver, because mRNA or proteins of these genes were exclusively expressed by periportal hepatocytes.     

Endogenous prostaglandin D(2) synthesis decreases vascular cell adhesion molecule-1 expression in human umbilical vein endothelial cells

We examined the role of prostaglandin D(2) (PGD(2)) in the expression of vascular cell adhesion molecule-1 (VCAM)-1 following interleukin-1beta (IL-1) stimulation in human umbilical vein endothelial cells (HUVEC) transfected with lipocaline-type PGD(2) synthase (L-PGDS) genes. HUVEC were isolated from human umbilical vein and incubated with 20 U/ml IL-1 and various concentrations of authentic PGD(2). The isolated HUVEC were also transfected with L-PGDS genes by electroporation. The L-PGDS-transfected HUVEC were used to investigate the role of endogenous PGD(2) in IL-1-stimulated VCAM-1 biosynthesis. We also used an anti-PGD(2) antibody to examine whether an intracrine mechanism was involved in VCAM-1 production. PGD(2) and VCAM-1 levels were determined by radio- and cell surface enzyme-immunoassay, respectively. VCAM-1 mRNA was assessed by RT-PCR. IL-1-stimulated VCAM-1 expression by HUVEC was dose-dependently inhibited by authentic PGD(2). L-PGDS gene-transfected HUVEC produced more PGD(2) than HUVEC transfected with the reporter gene alone. IL-1 induced increases in VCAM-1 expression in HUVEC transfected with reporter genes alone. However, this effect was significantly attenuated in the case of IL-1 stimulation of HUVEC transfected with L-PGDS genes, and accompanied by an apparent suppression of VCAM-1 mRNA expression. Neutralization of extracellular PGD(2) by anti-PGD(2)-specific antibody influenced neither VCAM-1 mRNA expression nor VCAM-1 biosynthesis. In conclusion, HUVEC transfected with L-PGDS genes showed increased PGD(2) synthesis. This increase was associated with attenuation of both VCAM-1 expression and VCAM-1 mRNA expression. The results suggest that endogenous PGD(2) decreases VCAM-1 expression and VCAM-1 mRNA expression, probably through an intracrine mechanism.