Establishment and partial characterization of SV40 virus-immortalized hepatocyte lines of normal and lethal mutant mice carrying a deletion on chromosome 7

Deletions in chromosome 7 of the mouse have been shown to cause failure of expression of various hepatocyte-specific genes in newborn deletion homozygotes, including the gene encoding tyrosine amino transferase (TAT) (EC 2.6.1.5) (Gluecksohn-Waelsch, 1979). Primary liver cultures of newborn albino deletion mutant mice (c14CoS/c14CoS) and of phenotypically normal mice (c14CoS/cch or cch/cch) were infected with SV40 virus and multiplying hepatocytes selected in arginine-deficient medium containing epidermal growth factor (EGF), insulin, and hydrocortisone (HC). Resulting normal (NMH-ch) and mutant (NMH-m14) hepatocyte lines expressing integrated viral transforming sequences did not senesce, they multiplied autonomously of EGF in medium with insulin plus HC, and they retained hepatocyte-specific functions. Both lines synthesized arginine and contained albumin and alpha-fetoprotein (AFP) mRNAs. TAT-specific mRNA was detected in normal but not in mutant hepatocyte lines. A fragment of the mouse tyrosinase gene, known to map at the albino locus (c) within the region deleted in the c14CoS mutant, hybridized with a 2.5 kb EcoRI fragment of normal NMH-ch DNA, whereas this fragment was undetectable in mutant NMH-m14 DNA. These immortalized hepatocyte lines reflect important properties of normal and mutant liver tissues from which they were derived. The deletion mutant mouse cell lines may be useful for complementation studies involving sequences corresponding to the deletions that encode regulatory gene(s) involved in the control of inducible expression of certain hepatocyte-specific genes such as TAT.     

Hepatocyte cell lines established from transgenic mice harboring temperature-sensitive simian virus 40 large T-antigen gene.

To establish cell lines exhibiting differentiation phenotypes, the immortalized cell lines were rapidly established from the primary culture of different tissues of transgenic mice harboring SV40 temperature-sensitive large T-antigen gene. The established cell lines grew at permissive temperature (33 degrees C), but not at nonpermissive temperature (39 degrees C). Several different cell types could be rapidly immortalized and cloned from the adult transgenic mice tissues. Among those cell lines, the established hepatocyte cell lines (TLR cell lines) exhibited liver-specific morphological and biochemical properties, but their properties were not coupled with the growth condition modified by temperature. The hepatocyte cell lines showed an inducibility of P450IA1 by 3-methylcholanthrene as observed in rat livers and this liver-specific function was stable even after 6 months of culture by continuous passages.     

Tyrosine aminotransferase induced in cells genetically and epigenetically deficient for this enzyme

Mice homozygous for lethal deletions around the albino (c) locus are deficient for several liver-specific enzymes, including tyrosine aminotransferase (TAT). The structural gene coding for this enzyme appears to be intact in these mutants and can be “activated” in homozygous mutant mouse liver-rat hepatoma cell hybrids. The present study demonstrates that the mouse form of TAT can also be induced in both normal and mutant mouse skin-rat hepatoma cell hybrids. Thus, a liver-specific enzyme is expressed in skin cells, both normal and mutant, the normal differentiated state of which excludes TAT expression.     

Transcriptionally regulated immortalization overcomes side effects of temperature-sensitive SV40 large T antigen

The temperature-sensitive mutant of the SV40 virus large T antigen (TAg) tsA58 is frequently employed for the conditional immortalization of primary cells. By increasing the temperature to 39 degrees C, the activity of the mutant TAg is reduced and the status of such cells may then resemble more closely that of primary cells. As an alternative, we used a novel immortalization vector with a tetracycline-regulated expression of the wild-type TAg. This enabled us to investigate the effects of the immortalizing gene expression and of temperature shifts independently of each other. Even for wild-type TAg-derived cell lines the elevated temperatures led to various clone-dependent phenotypes. This suggests that in freshly established cell lines temperature-sensitive growth phenotypes can arise spontaneously and independently of a temperature-sensitive immortalizing gene. Similar effects were observed with spontaneously immortalized cells. On the other hand, not all of the ts-TAg-derived cell lines were proliferation arrested at the non-permissive temperature. Therefore, the assumption that temperature-sensitive growth is solely due to the ts-TAg must be verified for each ts-TAg-derived cell line individually. This complexity could be avoided by using the autoregulatory immortalization vector expressing the wild-type TAg.     

B cell development in mice that lack one or both immunoglobulin kappa light chain genes.

We have generated mice that lack the ability to produce immunoglobulin (Ig) kappa light chains by targeted deletion of J kappa and C kappa gene segments and the intervening sequences in mouse embryonic stem cells. In wild type mice, approximately 95% of B cells express kappa light chains and only approximately 5% express lambda light chains. Mice heterozygous for the J kappa C kappa deletion have approximately 2-fold more lambda+ B cells than wild-type littermates. Compared with normal mice, homozygous mutants for the J kappa C kappa deletion have about half the number of B cells in both the newly generated and the peripheral B cell compartments, and all of these B cells express lambda light chains in their Ig. Therefore, homozygous mutant mice appear to produce lambda-expressing cells at nearly 10 times the rate observed in normal mice. These findings demonstrate that kappa gene assembly and/or expression is not a prerequisite for lambda gene assembly and expression. Furthermore, there is no detectable rearrangement of 3' kappa RS sequences in lambda+ B cells of the homozygous mutant mice, thus rearrangements of these sequences, per se, is not required for lambda light chain gene assembly. We discuss these findings in the context of their implications for the control of Ig light chain gene rearrangement and potential applications of the mutant animals.     

Phosphoenolpyruvate carboxykinase is necessary for the integration of hepatic energy metabolism

We used an allelogenic Cre/loxP gene targeting strategy in mice to determine the role of cytosolic phosphoenolpyruvate carboxykinase (PEPCK) in hepatic energy metabolism. Mice that lack this enzyme die within 3 days of birth, while mice with at least a 90% global reduction of PEPCK, or a liver-specific knockout of PEPCK, are viable. Surprisingly, in both cases these animals remain euglycemic after a 24-h fast. However, mice without hepatic PEPCK develop hepatic steatosis after fasting despite up-regulation of a variety of genes encoding free fatty acid-oxidizing enzymes. Also, marked alterations in the expression of hepatic genes involved in energy metabolism occur in the absence of any changes in plasma hormone concentrations. Given that a ninefold elevation of the hepatic malate concentration occurs in the liver-specific PEPCK knockout mice, we suggest that one or more intermediary metabolites may directly regulate expression of the affected genes. Thus, hepatic PEPCK may function more as an integrator of hepatic energy metabolism than as a determinant of gluconeogenesis.     

Histochemical differences in expression of X-linked glucose-6-phosphate dehydrogenase between ectoderm- and endoderm-derived embryonic and extra-embryonic tissues

We examined the activity of X-linked glucose-6-phosphate dehydrogenase (G6PD) in concepti of the enzyme-deficient mutant and wild-type C3H mice. By using different crosses between the G6PD-deficient homozygous, heterozygous, or wild-type females and hemizygous or wild-type males, we confirmed the inactivation of one of the two X chromosomes in female concepti by a histochemical method. With this technique, a dual (G6PD + or -) cell population could be observed in the tissue sections. We demonstrate that the paternal X chromosome is inactivated in the endoderm of parietal and visceral yolk sac and in the trophoblast, whereas in the embryo and in the yolk sac mesoderm this inactivation is random. Our results confirm biochemical observations showing that only the maternal X chromosome is expressed in all derivatives of trophectoderm and primitive endoderm, whereas derivatives of the primitive ectoderm show random X chromosome expression.     

Regulators of fetal liver differentiation in vitro

Seventeen-day-old fetal rat hepatocytes were employed to examine factors required to promote differentiation in vitro. In the absence of effectors, primary fetal hepatocytes dedifferentiated, as characterized by the rapid decline in synthesis of fetal alpha-fetoprotein (AFP), albumin, and transferrin. On the other hand, cells maintained in the presence of glucocorticoid hormone produced high levels of albumin and transferrin. Glucocorticoid could not prevent the decline in fetal AFP synthesis, but induced synthesis of the 65K variant AFP--the major AFP species produced by adult rat liver. Fetal hepatocytes maintained in the presence of 8-bromo-cAMP (8-BrcAMP), or methyl isobutyl xanthine (MIX), an agent that increases intracellular cAMP levels, synthesized high levels of fetal AFP and albumin but reduced levels of transferrin. Both glucocorticoid and 8-BrcAMP or MIX induced expression of adult liver-specific genes such as tyrosine aminotransferase (TAT) and phosphoenolpyruvate carboxykinase (PEPCK), suggesting that these fetal hepatocytes have matured. Cells maintained in the presence of glucocorticoid hormone and MIX (or 8-BrcAMP) contained more albumin, TAT, and PEPCK mRNAs and synthesized increased amounts of the 65K variant AFP than those with either agent alone. However, the glucocorticoid/MIX cells produced intermediate levels of the fetal AFP and transferrin. Our data indicate that both glucocorticoid hormone and cAMP are necessary for optimal differentiation of fetal hepatocytes in vitro.     

Chromosomal assignment and trans regulation of the tyrosine aminotransferase structural gene in hepatoma hybrid cells.

The structural gene encoding liver-specific tyrosine aminotransferase (TAT; EC 2.6.1.5) was assigned to mouse chromosome 8 by screening a series of hybrid cell lines for retention of murine Tat-1 gene sequences by genomic Southern blotting. This assignment demonstrated that the Tat-1 structural gene was not syntenic with Tse-1, a chromosome 11-linked locus that negatively regulates TAT expression in trans (A. M. Killary and R. E. K. Fournier, Cell 38:523-534, 1984). We also showed that the fibroblast Tat-1 gene was systematically activated in hepatoma X fibroblast hybrids retaining fibroblast chromosomes 8 in the absence of chromosome 11 but was extinguished in cells retaining both fibroblast chromosomes. Thus, the TAT structural genes of both parental cell types were coordinately regulated in the intertypic hybrids, and the TAT phenotype of the cells was determined by the presence or absence of fibroblast Tse-1.     

Functional annotation of mouse mutations in embryonic stem cells by use of expression profiling

Expression profiling offers a potential high-throughput phenotype screen for mutant mouse embryonic stem (ES) cells. We have assessed the ability of expression arrays to distinguish among heterozygous mutant ES cell lines and to accurately reflect the normal function of the mutated genes. Two ES cell lines hemizygous for overlapping regions of mouse Chromosome (Chr) 5 differed substantially from the wildtype parental line and from each other. Expression differences included frequent downregulation of hemizygous genes and downstream effects on genes mapping to other chromosomes. Some genes were affected similarly in each deletion line, consistent with the overlap of the deletions. To determine whether such downstream effects reveal pathways impacted by a mutation, we examined ES cell lines heterozygous for mutations in either of two well-characterized genes. A heterozygous mutation in the gene encoding the cell cycle regulator, cyclin D kinase 4 (Cdk4), affected expression of many genes involved in cell growth and proliferation. A heterozygous mutation in the ATP binding cassette transporter family A, member 1 (Abca1) gene, altered genes associated with lipid homeostasis, the cytoskeleton, and vesicle trafficking. Heterozygous Abca1 mutation had similar effects in liver, indicating that ES cell expression profile reflects changes in fundamental processes relevant to mutant gene function in multiple cell types.     

Gene deletion reveals roles for annexin A1 in the regulation of lipolysis and IL-6 release in epididymal adipose tissue

In this study, epididymal adipose tissue from male annexin 1 (ANXA1)-null and wild-type control mice were used to explore the potential role of ANXA1 in adipocyte biology. ANXA1 was detected by Western blot analysis in wild-type tissue and localized predominantly to the stromal-vascular compartment. Epididymal fat pad mass was reduced by ANXA1 gene deletion, but adipocyte size was unchanged, suggesting that ANXA1 is required for the maintenance of adipocyte and/or preadipocyte cell number. Epididymal tissue from wild-type mice responded in vitro to noradrenaline and isoprenaline with increased glycerol release, reduced IL-6 release, and increased cAMP accumulation. Qualitatively similar but significantly attenuated responses to the catecholamines were observed in tissue from ANXA1-null mice, an effect that was not associated with changes in beta-adrenoceptor mRNA expression. Lipopolysaccharide (LPS) also stimulated lipolysis in vitro, but its effects were muted by ANXA1 gene deletion. By contrast, LPS failed to influence IL-6 release from wild-type tissue but stimulated the release of the cytokine from tissue from ANXA1-null mice. ANXA1 gene deletion did not affect glucocorticoid receptor expression or the ability of dexamethasone to suppress catecholamine-induced lipolysis. It did, however, augment IL-6 expression and modify the inhibitory effects of glucocorticoids on IL-6 release. Collectively, these studies suggest that ANXA1 supports aspects of adipose tissue mass and alters the sensitivity of epididymal adipose tissue to catecholamines, glucocorticoids, and LPS, thereby modulating lipolysis and IL-6 release.