Large scale gene expression analysis of CBA/J mouse strain fetal thymus using cDNA-array hybridizations

The CBA/J inbred mouse strain constitutes an interesting in vivo model-system for studies on molecular genetics of thymus ontogeny. Using RT-PCR method we have found previously that several immune system related genes as interleukins and MHC are differentially expressed. During this period the onset of T-cell receptor beta rearrangements also occur. To know which other genes are modulated during the ontogeny of the thymus, the mRNA expression levels of fetal thymus (15 and 16 days gestation) of CBA/J mouse strain were measured by hybridization with a set of four macroarrays containing a panel of 6,144 IMAGE cDNA clones from MTB thymus library. We found 145 differentially expressed sequences; 44 were up- and 101 down-regulated in the thymus at 15–16 days gestation. Among these sequences, only 20 are identified as genes whose functions are known and 125 are still unknown. Our data demonstrated that, despite intense research on maturation of the immune system focusing on the activity of several well-characterized genes, the large scale expression profile during thymus ontogeny is still an open matter. The use of cDNA-array technology is an affordable method to identify new genes that may play a role in this phenomenon.     

Single nucleotide polymorphism (SNP) discovery in porcine expressed genes

High-throughput genotyping of swine populations is a potentially efficient method for establishing animal lineage and identification of loci important to animal health and efficient pork production. Markers were developed based upon single nucleotide polymorphisms (SNPs), which are abundant and amenable to automated genotyping platforms. The focus of this research was SNP discovery in expressed porcine genes providing markers to develop the porcine/human comparative map. Locus specific amplification (LSA) and comparative sequencing were used to generate PCR products and allelic information from parents of a swine reference family. Discovery of 1650 SNPs in 403 amplicons and strategies for optimizing LSA-based SNP discovery using alternative methods of PCR primer design, data analysis, and germplasm selection that are applicable to other populations and species are described. These data were the first large-scale assessment of frequency and distribution of porcine SNPs.     

Mapping microsatellite markers identified in porcine EST sequences

A sequence search of swine expressed sequence tags (EST) data in GenBank identified over 100 sequence files which contained a microsatellite repeat or simple sequence repeat (SSR). Most of these repeat motifs were dinucleotide (CA/GT) repeats; however, a number of tri-, tetra-, penta- and hexa-nucleotide repeats were also detected. An initial assessment of six dinucleotide and 14 higher-order repeat markers indicated that only dinucleotide markers yielded a sufficient number of informative markers (100% vs. 14% for dinucleotide and higher order repeats, respectively). Primers were designed for an additional 50 di- and one tri-nucleotide SSRs. Overall, 42 markers were polymorphic in the US Meat Animal Research Center (MARC) reference population, 17 markers were uninformative and 12 primer pairs failed to satisfactorily amplify genomic DNA. A comparison of di-nucleotide repeat vs. markers with repeat motifs of three to six bases demonstrated that 72% of dinucleotide markers were informative relative to only 7% of other repeat motifs. The difference was the result of a much higher percentage of monomorphic markers in the three to six base repeat motif markers than in the dinucleotide markers (64% vs. 14%). Either higher order repeat motifs are less polymorphic in the porcine genome or our selection criteria for repeat length of more than 17 contiguous bases was too low. The mapped microsatellite markers add to the porcine genetic map and provide valuable links between the porcine and human genome.     

Kinetics of thymocyte developmental process in fetal and neonatal mice

Kinetics of thymocyte development in vivo during embryogenesis was pursued. The early development of thymocytes in the fetal and neonatal BALB/c mice was discontinuous, with four waves of cell proliferation occurring at fetal day (Fd) 14 to 17, Fd 18 to day (D) 1 after birth, D 2 to D 5 and D6 thereafter. The first three proliferation waves coincided with the generation of CD4^hiCD8^hi (DP), TCR CD4^hiCD8^-/^loCD8^int/hi(CD4 SP), and TCR CD4^-/^loCD8^int/hi (CD8 SP) thymocytes, respectively. The transition from DN to DP cells was further investigated and it was found out that there were two differential pathways via im-mature single positive (ISP) cells in the BALB/c mice, each functioning at different fetal ages. One is via TCR^-CD4^-CD8^ cells, occurring between Fd 15 and Fd 17 and the other is via TCR^-CD4^ CD86-cells,occurring from Fd 17 until birth. In contrast, the TCR^-CD4^-CD8^ pathway dominated overwhelminglyin the C57BL/6 mice. These findings shed new light on the hypothesis that the differential pathway pref-erence varies with mouse strains. With respect to the shift in the intensity of CD4 and CD8 expression onthymocytes from fetal to adult mice, the TCR CD4^hiCD8^-/^lo, and TCR^ CD4^-/^loCD8^int/hi subsets might be equivalent to the medullary type TCR^ CD4/CD8 SP cells.     

Gene expression profiling between embryonic and larval stages of the silkworm, Bombyx mori

To elucidate the molecular mechanisms associated with metamorphic phenomenon relating to Bombyx mori, an important organism in the sericulture industry, we identified genes that are expressed in the different developmental stages, specifically the embryonic (ES) and larval (LS) stages of B. mori. Of 8230 high-quality ESTs from two full-length enriched cDNA libraries, 3442 of the ES ESTs were coalesced into 1325 clusters, while 4788 were coalesced into 927 clusters. The functional classification of these ESTs based on Gene Ontology showed that the types of genes that are associated with oxidoreductase activity, enzyme inhibition, and larval development were highly observed in LS, whereas the types of genes that are involved in nucleotide binding, enzyme activity, and protein transport activity were highly observed in ES. In addition, when the gene expression profile between ES and LS was examined by counting the EST frequencies in each library, 69 genes were identified as being either up- or down-regulated in the larval stage compared to the embryonic stage (P>0.99) and this was confirmed by semi-quantitative RT-PCR. The results show that genes involved in proteolysis and peptidolysis, and lipid and carbohydrate metabolism were dramatically up-regulated in LS, while those related to protein metabolism, DNA/RNA, and coenzymes were highly down-expressed. In particular, a GO analysis of these genes revealed that genes that are involved in hydrolase activity were observed to be highly expressed in amount as well as diversity in LS, while those involved in nucleic acid binding were highly expressed in ES. These data may contribute to elucidating genetic events that distinguish the developmental stage and to our understanding of the metamorphosis of B. mori.     

Analysis of cellular heterogeneity in mouse thymus cultures

Summary Analysis of 5 to 6 d primary cultures of cells derived from murine thymus glands revealed a heterogeneous population of cells rather than “pure” reticuloepithelial cell cultures as was assumed previously by other investigators. The monolayer cultures consisted of at least three cell types: thymus epithelial cells, macrophagelike epithelioid cells, and fibroblasts. Surprisingly, about 50% of the cells had positive cytochemical staining reactions for acid phosphatase and nonspecific esterase. The same cells phagocytized carbon particles, latex beads, and yeast. Furthermore, these cells could be removed from the initial cell suspension by phagocytosis of carbonyl iron, followed by magnetic separation, but once they had adhered to the substratum they were resistant to trypsin removal. All of these findings supported the conclusion that about 50% of the cells in the monolayers were macrophages. The other cells present were thymus epithelial cells and a small number of fibroblasts. Both of the latter types of cell were cytochemically negative, did not phagocytize particulate material, and were not removed by carbonyl iron treatment, but were removed by treating the monolayer with trypsin. The findings in this report indicated that epithelioid morphology alone was inadequate to identify correctly the cell types found in thymus cultures and that the use of such cultures as a model to study in vitro the maturation of certain immunological functions has been based on assumptions here shown to be incorrect. This work was supported by the Graduate School, The Ohio State University, the Bremer Foundation, the American Cancer Society (IN-16R), and National Cancer Institute Grant 5 ROL CA-19346-03.     

Gene expression profiling in porcine mammary gland during lactation and identification of breed-and developmental-stage-specific genes

A total of 28941 ESTs were sequenced from five 5′-directed non-normalized cDNA libraries, which were assembled into 2212 contigs and 5642 singlets using CAP3. These sequences were annotated and clustered into 6857 unique genes, 2072 of which having no functional annotations were considered as novel genes. These genes were further classified into Gene Ontology categories. By comparing the expression profiles, we identified some breed-and developmental-stage-specific gene groups. These genes may be relative to reproductive performance or play important roles in milk synthesis, secretion and mammary involution. The unknown EST sequences and expression profiles at different developmental stages and breeds are very important resources for further research.     

Gene expression profiling in porcine mammary gland during lactation and identification of breed-and developmental-stage-specific genes

The mammary gland provides an excellent system to study questions pertaining to organogenesis, cell differentiation and oncogenesis. Intensive efforts have been made to understand the development of the mammary gland, particularly in terms of lactogenesis…     

不同电转仪的电转参数、质粒用量和拓扑结构对猪胎儿成纤维细胞转染效率的影响

为获得猪胎儿成纤维细胞(porcine fetal fibroblasts, PFFs)最佳的电转染效率,本研究利用荧光激活细胞分选技术(fluorescence activated cell sorting, FACS)辅助优化NEPA 21和Nucleofector? 2b两种电转仪电转染PFFs细胞的参数,比较不同质粒用量和拓扑结构在ECM^? 830、NEPA 21和Nucleofector? 2b中的转染效率。结果显示：NEPA 21电转PFFs的最佳穿孔参数为脉冲电压200 V,脉冲长度3 ms,脉冲间隔50 ms,脉冲次数3次,脉冲电压衰减幅度10%;Nucleofector? 2b在U-023的转染参数下达到最高转染效率。ECM^? 830和Nucleofector? 2b的最适质粒用量都为10 μg,而NEPA 21为8 μg;超螺旋质粒比线性化质粒的转染效率更高,且3种仪器中Nucleofector? 2b转染效果最佳。本研究综合考虑电转仪、电转参数、质粒用量和拓扑结构的影响因素以优化PFFs的电转条件,为高效制备转基因猪及基因编辑猪的研究奠定基础。     

Exogenous control of I-A expression in fetal thymus explants

With the use of a system in which 14 day fetal thymus lobes were cultured in vitro with bone marrow or other lymphoid cells, evidence was obtained that entry of macrophage/dendritic cells (M phi/DC) into the thymus causes a marked rise in the density of endogenous I-A molecules expressed in the cortex, presumably on epithelial cells. High cortical I-A expression also occurred when thymus lobes were cultured with supernatants containing IFN-gamma; addition of anti-IFN-gamma antibody blocked I-A induction. The working hypothesis for these findings is that cellular interactions occurring in the medullary region between M phi/DC and a subset of thymocytes leads to production of IFN-gamma, which then diffuses into the cortex and promotes epithelial I-A expression. The possible relevance of this scheme to the process of thymic "education" is discussed.     

Tetracycline-inducible gene expression in nuclear transfer embryos derived from porcine fetal fibroblasts transformed with retrovirus vectors

A critical problem of transgenic livestock production is uncontrollable constitutive expression of the foreign gene, which usually results in serious physiological disturbances in transgenic animals. One of the best solutions for this problem may be use of controllable gene expression system. In this study, using retrovirus vectors designed to express the enhanced green fluorescent protein (EGFP) gene under the control of the tetracycline-inducible promoter, we examined whether the expression of the transgene could be controllable in fibroblast cells and nuclear transfer (NT) embryos of porcine. Transformed fibroblast cells were cultured in medium supplemented with or without doxycycline (a tetracycline analog) for 48 hr, and the induction efficiency was measured by comparing EGFP gene expression using epifluorescence microscopy and Western and Northern blot analyses. After the addition of doxycycline, EGFP expression increased up to 17-fold. The nuclei of transformed fibroblast cells were transferred into enucleated oocytes. Fluorescence emission data revealed strong EGFP gene expression in embryos cultured with doxycycline, but little or no expression in the absence of the antibiotic. Our results demonstrate the successful regulation of transgene expression in porcine nuclear transfer embryos, and support the application of an inducible expression system in transgenic pig production to solve the inherent problems of side-effects due to constitutive expression of the transgene.     

毒品成瘾与脑组织基因表达谱的研究进展

毒品成瘾是由滥用毒品外在因素与遗传易感性等内在因素共同作用而导致的一种慢性脑疾病; 毒品成瘾的机制目前还不十分清楚。毒品成瘾研究的一个主要目标是鉴别和分离毒品导致脑功能障碍的分子机制; 采用高通量的基因表达谱技术研究毒品成瘾者在不同状态下脑基因表达全貌, 对深入认识毒品成瘾的机制具有重要的意义。文章综述了毒品成瘾的遗传机制及高通量脑组织基因组表达技术——SAGE和微阵列(Microarry)在毒品成瘾研究中的进展。