Muscle-specific gene expression controlled by a regulatory element lacking a MyoD1-binding site

Muscle-specific expression of the gene encoding the delta subunit of the acetylcholine receptor is controlled by a 54-base-pair region that does not contain a binding site for MyoD1, a protein involved in activation of the myogenic program. A MyoD1-binding site is present in the proximal promoter region of the gene encoding the delta-subunit, but is neither sufficient nor necessary for muscle-specific expression in transfected muscle cells.     

Expression of two myogenic regulatory factors myogenin and MyoD1 during mouse embryogenesis

MyoD1 and myogenin are muscle-specific proteins which can convert non-myogenic cells in culture to differentiated muscle fibres, implicating them in myogenic determination. The pattern of expression of MyoD1 and myogenin during the early stages of muscle formation in the mouse embryo in vivo and in limb-bud explants cultured in vitro, indicates that they may have different functions in different types of muscle during development.     

Localization of muscle gene products in nuclear domains

The localization of gene products is central to the development of cell polarity and pattern specification during embryogenesis. To monitor the distribution of gene products encoded by different nuclei in the same cell in tissue culture, we fused cells of different species to form multinucleated non-dividing heterokaryons. In previous fusion studies, cell-surface antigens and organelles contributed by disparate cell types intermixed within minutes. Using heterokaryons produced with differentiated muscle cells, we demonstrate here that a muscle membrane component, the Golgi apparatus mediating its transport, and a sarcomeric myosin heavy chain are localized in the vicinity of the nuclei responsible for their synthesis. These results provide direct evidence that products (organelle, membrane and structural proteins) derived from individual nuclei can remain localized in myotubes, a finding with implications both for neuromuscular synapse formation and for the carrier state of Duchenne muscular dystrophy.     

Production of transgenic rabbits, sheep and pigs by microinjection

Direct microinjection has been used to introduce foreign DNA into a number of terminally differentiated cell types as well as embryos of several species including sea urchin, Candida elegans, Xenopus, Drosophila and mice. Various genes have been successfully introduced into mice including constructs consisting of the mouse metallothionein-I (MT) promoter/regulator region fused to either the rat or human growth hormone (hGH) structural genes. Transgenic mice harbouring such genes commonly exhibit high, metal-inducible levels of the fusion messenger RNA in several organs, substantial quantities of the foreign growth hormone in serum and enhanced growth. In addition, the gene is stably incorporated into the germ line, making the phenotype heritable. Because of the scientific importance and potential economic value of transgenic livestock containing foreign genes, we initiated studies on large animals by microinjecting the fusion gene, MT-hGH, into the pronuclei or nuclei of eggs from superovulated rabbits, sheep and pigs. We report here integration of the gene in all three species and expression of the gene in transgenic rabbits and pigs.     

Differentiation of bone mar-row derived Thy-1~+β_2M~- cells into hepatocytes induced by coculture with transgenic CFSCs

Studies of transplantation in vivo indicted thatbone marrow derived stem cells had a potential to differenti-ate into mature hepatocytes. However, there are lots ofdoubts and uncertainties in the influencing factors and con-trol agents of effectively inducing stem cell differentiation invitro, the efficiency of stem cells‘ differentiation into hepato-cytes and differentiated cells‘ life-span and functional state,etc. In this study, rat bone marrow derived Thy-l^ β2M^- cells(BDTCs) were induced to differentiate into hepatocytes byco-culturing with CFSC/HGF feeder layers which expressedhHGF efficiently and stably. RT-PCR and immunofluores-cent texts proved induced BDTCs expressed infant and adulthepatocyte specific genes. Further more, these cells displayedfunctions of indocyanine green (ICG) uptake, ammoniummetabolism and albumin production. It was shown thatgrowth factors together with hepatic nonparenchyma cellsprovided a feasible microenvironment for differentiation ofbone marrow stem cells into hepatocytes. The studies notonly provided a significant biological model for going deepinto the mechanism of stem cell plasticity, but also offered atheoretical and technical foundation of gene and stem cellengineering-based regenerative medicine for end-stage liverdiseases.     

Nanog promotes transfer of pluripotency after cell fusion

Through cell fusion, embryonic stem (ES) cells can erase the developmental programming of differentiated cell nuclei and impose pluripotency. Molecules that mediate this conversion should be identifiable in ES cells. One candidate is the variant homeodomain protein Nanog, which has the capacity to entrain undifferentiated ES cell propagation. Here we report that in fusions between ES cells and neural stem (NS) cells, increased levels of Nanog stimulate pluripotent gene activation from the somatic cell genome and enable an up to 200-fold increase in the recovery of hybrid colonies, all of which show ES cell characteristics. Nanog also improves hybrid yield when thymocytes or fibroblasts are fused to ES cells; however, fewer colonies are obtained than from ES x NS cell fusions, consistent with a hierarchical susceptibility to reprogramming among somatic cell types. Notably, for NS x ES cell fusions elevated Nanog enables primary hybrids to develop into ES cell colonies with identical frequency to homotypic ES x ES fusion products. This means that in hybrids, increased Nanog is sufficient for the NS cell epigenome to be reset completely to a state of pluripotency. We conclude that Nanog can orchestrate ES cell machinery to instate pluripotency with an efficiency of up to 100% depending on the differentiation status of the somatic cell.     

Specific expression of an elastase-human growth hormone fusion gene in pancreatic acinar cells of transgenic mice

Transfection of genes into tissue culture cell lines has demonstrated that relatively short DNA sequences can allow expression of immunoglobulin, insulin and chymotrypsin genes in their appropriate cell types. A definitive test of cell-specific gene expression, however, requires testing genes in every possible cell type, an experiment performed easily by introducing the gene in question into the germ line of an animal. Transfer of intact genes into mice has demonstrated that a mouse immunoglobulin kappa gene is expressed specifically in B lymphocytes, a rat elastase I gene is expressed specifically in pancreas and a chicken transferrin gene is expressed preferentially in liver. Mouse metallothionein-growth hormone fusion genes introduced into mice are preferentially expressed in the liver, consistent with the expression of endogenous metallothionein genes, but initial experiments with beta-globin genes have not revealed proper regulation. To identify the DNA elements required for pancreas-specific expression of the rat elastase I gene, we joined the 5'-flanking region of this gene to the human growth hormone (hGH) structural gene and introduced the fusion gene into mice. Here we demonstrate that a fusion gene containing only 213 base pairs (bp) of elastase I gene sequence directs expression of hGH in pancreatic acinar cells.     

大鼠再生肝8种细胞的嘌呤核苷酸代谢基因转录谱预示的代谢活动

为了解大鼠肝再生中肝细胞、胆管上皮细胞、卵圆细胞、星形细胞、窦内皮细胞、库普弗细胞、陷窝细胞、树突状细胞等8种肝脏细胞的嘌呤核苷酸代谢基因转录谱及预示的代谢活动,按张丽君[1]等方法分离大鼠部分肝切除后10个恢复时间点大鼠再生肝的上述8种细胞,用RatGenome2302.0芯片等检测嘌呤核苷酸代谢基因在上述细胞中表达变化,用Excel等软件及生物信息学和系统生物学等方法分析它们的表达模式、预示的生理活动等.结果表明,85个嘌呤核苷酸代谢基因在大鼠肝再生中发生了有意义表达变化,8种细胞的相应基因数为40,43,30,42,22,26,36和48.上调、下调、上/下调的基因个数为49、11、31,相应细胞的基因数为27、20和1,31、4和1,15、7和3,12、10和0,23、15和3,19、7和2,39、3和1,33、6和0.其中,催化DNA合成的DNA聚合酶基因和催化RNA合成的RNA聚合酶基因在肝再生的多个时间点和多种细胞中表达增强,胆管上皮细胞和星形细胞的腺苷酸合成相关基因表达增强.肝细胞、卵圆细胞和星形细胞的核苷酸分解相关基因、肝细胞、星形细胞和树突状细胞的嘌呤核苷分解相关基因表达减弱.预示大鼠肝再生与嘌呤核苷酸代谢密切相关.     

Transplanted bone marrow regenerates liver by cell fusion

Results from several experimental systems suggest that cells from one tissue type can form other tissue types after transplantation. This could be due to the presence of multipotential or several types of adult stem cells in donor tissues, or alternatively, to fusion of donor and recipient cells. In a model of tyrosinaemia type I, mice with mutations in the fumarylacetoacetate hydrolase gene (Fah-/-) regain normal liver function after transplantation of Fah+/+ bone marrow cells, and form regenerating liver nodules with normal histology that express Fah. Here we show that these hepatic nodules contain more mutant than wild-type Fah alleles, and that their hepatocytes express both donor and host genes, consistent with polyploid genome formation by fusion of host and donor cells. Using bone marrow cells marked with integrated foamy virus vectors that express green fluorescent protein, we identify common proviral junctions in hepatic nodules and haematopoietic cells. We also show that the haematopoietic donor genome adopts a more hepatocyte-specific expression profile after cell fusion, as the wild-type Fah gene was activated and the pan-haematopoietic CD45 marker was no longer expressed.     

Stable expression of two variable surface glycoproteins by cloned Trypanosoma equiperdum

African trypanosomes are thought to evade the host immune system by periodically changing their variable surface glycoprotein (VSG). VSG genes are activated by a complex process involving the duplicative transposition of silent basic copy genes to one of several expression sites. These expression-linked copies (ELCs) of the VSG genes are also subject to regulation within expression sites by as yet unknown mechanisms. It is generally assumed that trypanosomes can express only one VSG gene at a time. Nevertheless, the finding that they contain multiple VSG gene expression sites suggests that multiple expression is possible. We show here that Trypanosoma equiperdum can stably express two VSG genes in a simple axenic culture system and that both antigens are present on the cell surface. The two antigens do not co-cap or form heterodimers. Their corresponding genes show no cross-hybridization and are situated in different telomere-linked expression sites. Northern blot analysis reveals that both genes are active in the double expressors.