Comparison of gene expression during in vivo and in vitro postnatal retina development

Retina explants are widely used as a model of neural development. To define the molecular basis of differences between the development of retina in vivo and in vitro during the early postnatal period, we carried out a series of microarray comparisons using mouse retinas. About 75% of 8,880 expressed genes from retina explants kept the same expression volume and pattern as the retina in vivo. Fewer than 6% of the total gene population was changed at two consecutive time points, and only about 1% genes showed more than a threefold change at any time point studied. Functional Gene Ontology (GO) mapping for both changed and unchanged genes showed similar distribution patterns, except that more genes were changed in the GO clusters of response to stimuli and carbohydrate metabolism. Three distinct expression patterns of genes preferentially expressed in rod photoreceptors were observed in the retina explants. Some genes showed a lag in increased expression, some showed no change, and some continued to have a reduced level of expression. An early downregulation of cyclin D1 in the explanted retina might explain the reduction in numbers of precursors in explanted retina and suggests that external factors are required for maintenance of cyclin D1. The global view of gene profiles presented in this study will help define the molecular changes in retina explants over time and will provide criteria to define future changes that improve this model system.     

Lentivirus vector purification using anion exchange HPLC leads to improved gene transfer

Recombinant lentiviral vectors stably transduce both dividing and nondividing cells. Virus pseudotyping with vesicular stomatitis virus envelope G (VSV-G) protein broadens the host range of lentiviral vector and enables vector concentration by ultra-centrifugation. However, as a result of virus vector concentration, contaminating protein debris derived from vector-producing cell culture media is toxic to target cells and reduces the transduction efficiency. Here we report a new and rapid technique for purifying lentivirus vector using the strong anion exchange column that significantly improves gene transfer rates. We purified VSV-G pseudotyped self-inactivating lentivirus vector and obtained two protein elution peaks (Peak 1 and Peak 2) corresponding to transducing activity. Peak 1 viral particles were 4-8 times more effective in transducing target cells than Peak 2 or non-purified (pre-HPLC) viral particles. We used purified lentivirus vector expressing the human Fanconi anemia group A (FANCA) gene to transduce murine hematopoietic stem/progenitor cells. We observed a consistent 2- to 3-fold increase in gene transfer rates using Peak 1 purified virus compared with non-purified virus. We conclude that the purification method using the HPLC system provides the highly purified virus vector that reduces cell toxicity and significantly improves gene transfer in primary cells.     

Lentivirus vector-mediated hematopoietic stem cell gene transfer of common gamma-chain cytokine receptor in rhesus macaques

Nonhuman primate model systems of autologous CD34+ cell transplant are the most effective means to assess the safety and capabilities of lentivirus vectors. Toward this end, we tested the efficiency of marking, gene expression, and transplant of bone marrow and peripheral blood CD34+ cells using a self-inactivating lentivirus vector (CS-Rh-MLV-E) bearing an internal murine leukemia virus long terminal repeat derived from a murine retrovirus adapted to replicate in rhesus macaques. In vitro cytokine stimulation was not required to achieve efficient transduction of CD34+ cells resulting in marking and gene expression of the reporter gene encoding enhanced green fluorescent protein (EGFP) following transplant of the CD34+ cells. Monkeys transplanted with mobilized peripheral blood CD34+ cells resulted in EGFP expression in 1 to 10% of multilineage peripheral blood cells, including red blood cells and platelets, stable for 15 months to date. The relative level of gene expression utilizing this vector is 2- to 10-fold greater than that utilizing a non-self-inactivating lentivirus vector bearing the cytomegalovirus immediate-early promoter. In contrast, in animals transplanted with autologous bone marrow CD34+ cells, multilineage EGFP expression was evident initially but diminished over time. We further tested our lentivirus vector system by demonstrating gene transfer of the human common gamma-chain cytokine receptor gene (gamma(c)), deficient in X-linked SCID patients and recently successfully used to treat disease. Marking was 0.42 and.001 HIV-1 vector DNA copy per 100 cells in two animals. To date, all EGFP- and gamma(c)-transplanted animals are healthy. This system may prove useful for expression of therapeutic genes in human hematopoietic cells.     

Differential expression of genes involved in cGMP-dependent nitric oxide signaling in murine embryonic stem (ES) cells and ES cell-derived cardiomyocytes.

Nitric oxide (NO) performs multiple physiological roles as a biological signaling molecule. The role of NO and cGMP signaling in embryonic stem (ES) cell-derived cardiomyocytes (CM) has been investigated but many questions remain. In this study, we examined the expression of the NO signaling pathway components nitric oxide synthase (NOS-1, 2, 3), soluble guanylyl cyclase (sGCalpha(1) and beta(1)) and protein kinase G (PKG) genes and sGC activity in murine ES cells subjected to differentiation by embryoid body (EB) formation. We found that in undifferentiated ES cells, NOS-1, NOS-3, and sGCbeta(1) were detected while NOS-2, sGCalpha(1), and PKG were very low or undetectable. When ES cells were subjected to differentiation, NOS-1 abruptly decreased within one day, NOS-2 mRNA became detectable after several days, and NOS-3 increased after 7-10 days. Levels of sGCalpha(1), sGCbeta(1), and PKG all increased gradually over a several day time course of differentiation in EB outgrowths. Analysis of sGC activity in cell lysates derived from undifferentiated ES cells revealed that NO could not stimulate cGMP. However, lysates from differentiated EB outgrowths produced abundant cGMP levels after NO stimulation. Purification of ES-cell derived CM revealed that mRNA expression of all the NOS isoforms was very low to absent while sGCalpha(1) and beta(1) subunit mRNAs were abundant and sGC-mediated cGMP production was apparent in this population of cells. These data suggest that cGMP-mediated NO signaling may play a minor role, if any, in undifferentiated ES cells but could be involved in the early differentiation events or physiological processes of ES cells or ES cell-derived lineages.     

Light-regulated gene expression during maize leaf development

We have established schedules of expression during maize leaf development in light and darkness for the messenger RNAs (mRNAs) and polypeptides for ribulose 1,5-bisphosphate carboxylase (RuBPCase) subunits, phosphoenolpyruvate carboxylase (PEPCase), and the light- harvesting chlorophyll a/b-binding protein (LHCP). Levels of mRNAs were measured by hybridization with cloned probes, and proteins were measured by immunodetection on protein gel blots. The initial synthesis in leaves of all four mRNAs follows a light-independent schedule; illumination influences only the level to which each mRNA accumulates. The synthesis of RuBPCase small and large subunits and of PEPCase polypeptides also follows a light-independent schedule which is modified quantitatively by light. However, the accumulation of LHCP polypeptides absolutely requires illumination. The accumulation of each protein closely follows the accumulation of its mRNA during growth in light. Higher ratios of PEPCase and RuBPCase protein to mRNA occur during dark growth.     

Patterns of angiogenic and hematopoietic gene expression during brown trout embryogenesis

In this article, whole mount in situ hybridization is used to examine early blood vessel and blood cell development in the embryos of the brown trout Salmo trutta lacustris. cDNAs encoding for the angiogenic markers fli1 and flk1, and for the hematopoietic markers gata1 and gata2, were identified from an expressed sequence tag library of rainbow trout. Results show that fli1, flk1 and gata2 are activated in bilateral bands of the lateral trunk mesoderm before the onset of somitogenesis, shortly followed by gata1. These bands then converge toward the ventral midline to form the intermediate cell mass (ICM) (anterior ICM). Subsequent axial vasculogenesis and initial blood cell formation involve a clear spatial separation of fli1 and gata gene expression. Fli1 staining is most intense within the axial vessel (dorsal aorta, posterior cardinal vein) forming and lateral ICM cells, whereas binding of gata1 and gata2 probes becomes confined to the central portion of ICM cells beneath the dorsal aorta. This is followed by a first wave of angiogenesis, indicated by expression of fli1 and flk1. This gives rise to the intersegmental, dorsal longitudinal anastomotic and intestinal vessels. Further angiogenesis and hematopoiesis are activated in the "posterior ICM" of the tail. Here, the absence of gata1 indicates that hematopoiesis in this tissue generates myeloid rather than erythroid cells. The results supplement and validate previous, now historical morphological work in salmonids, thus aiding the elucidation of a comprehensive general scheme of angiogenic and hematopoietic development in the teleost embryo.     

Lithium influences differentiation and tissue-specific gene expression of mouse embryonic stem (ES) cells in vitro

The effects of lithium chloride (LiCl) on differentiation of mouse embryonic stem (ES) cells were investigated in order to evaluate the ES cell test (EST) used in a European Union validation study for screening of embryotoxic agents in vitro. We show that LiCl inhibited concentration-dependently the differentiation of ES cells into cardiac and myogenic cells. Whereas the inhibition of cardiac differentiation by high concentrations of LiCl was obvious at day 5 + 5, decreased skeletal muscle cell differentiation was observed only at day 5 + 8. Semi-quantitative RT-PCR analyses revealed significantly lower levels of mRNA encoding cardiac-specific alpha-myosin heavy chain and skeletal muscle-specific myoD. By morphological investigation, an influence of lithium on neuronal differentiation was not evident. However, mRNA levels of genes encoding synaptophysin and the 160 kDa neurofilament protein were increased by high LiCl concentrations, whereas mRNA levels of mash-1 and Engrailed-1 were decreased, suggesting a specific influence of lithium on neuronal differentiation. Furthermore, LiCl treatment resulted in a slight, but non-significant increase of beta-catenin levels in ES cell-derived embryoid bodies. Our results demonstrate that the ES cell test, EST may be suitable to detect inhibitory effects of test compounds especially on cardiac differentiation, whereas effects on neuronal cells would not be detected. Therefore, we propose that morphological analyses of cardiac differentiation alone are insufficient to detect embryotoxic effects. The assay of other cell lineages at different developmental stages, and expression analyses of tissue-specific genes should also be employed.     

Polony analysis of gene expression in ES cells and blastocysts

Expression profiling of stem cells is challenging due to their small numbers and heterogeneity. The PCR colony (polony) approach has theoretical advantages as an assay for stem cells but has not been applied to small numbers of cells. An assay has been developed that is sensitive enough to detect mRNAs from small numbers of ES cells and from fractions of a single mouse blastocyst. Genes assayed include Oct3, Rex1, Nanog, Cdx2 and GLUT-1. The assay is highly sensitive so that multiple mRNAs from a single blastocyst were easily detected in the same assay. In its present version, the assay is an attractive alternative to conventional RT–PCR for profiling small populations of stem cells. The assay is also amenable to improvements that will increase its sensitivity and ability to analyze many cDNAs simultaneously.