Involvement of phosphoinositide 3-kinase signaling pathway in chondrocytic differentiation of ATDC5 cells: application of a gene-trap mutagenesis

Gene-trap mutagenesis is based on the notion that the random insertion of a trapping vector may disturb the function of inserted genes. Here, we applied this method to murine mesenchymal ATDC5 cells, which differentiate into mature chondrocytes in the presence of insulin. As the trap vector we used pPT1-geo, which lacks its own promoter and enhancer, but contains a lacZ-neo fusion gene as a reporter and selection marker driven by the promoter of the trapped gene. After pPT1-geo was introduced into ATDC5 cells by electroporation, the neomycin-resistant clones were screened for beta-galactosidase activity. The selected clones were cultured in differentiation medium to evaluate the chondrogenic phenotype. The clones no. 6-30 and 6-175, which exhibited impaired and accelerated mineralization, respectively, were subjected to further analysis. In clone no. 6-30 in which the gene coding for the p85alpha subunit of phosphoinositide 3-kinase (PI3K) was trapped, the expression of marker genes of early chondrocytes including collagen type II, aggrecan, and PTH/PTHrP receptor was delayed. The insulin-induced stimulation of growth was reduced in clone no. 6-30 compared with the parental ATDC5 cells. Moreover, treatment of parental ATDC5 cells with a specific inhibitor of PI3K, LY294002, phenocopied clone no. 6-30, suggesting the involvement of PI3K signaling in the chondrogenic differentiation of ATDC5 cells. Clone no. 6-175 with accelerated mineralization was revealed to have a gene homologous to human KIAA0312 trapped, whose function remains unclear. Taken together, the gene-trap in ATDC5 cells might be useful to identify the molecules involved in chondrogenic differentiation.     

Isolation of cDNA and genomic DNA clones encoding type II collagen.

A cDNA library constructed from total chick embryo RNA was screened with an enriched fraction of type II collagen mRNA. Two overlapping cDNA clones were characterized and shown to encode the COOH propeptide of type II collagen. In addition, a type II collagen clone was isolated from a Charon 4A library of chick genomic fragments. Definitive identification of the clones was based on DNA sequence analysis. The 3' end of the type II collagen gene appears to be similar to that of other interstitial collagen genes. Northern hybridization data indicates that there is a marked decrease in type II collagen mRNA levels in chondrocytes treated with the dedifferentiating agent 5-bromodeoxyuridine. The major type II collagen mRNA species is 5300 bases long, similar to that of other interstitial collagen RNAs.     

A short repeat in the genome of the DNA sequences flanking bovine growth hormone genes

The phage clones containing a gene coding for bovine growth hormone were isolated from a bovine genomic library. Comparison of the 5' and 3' regions flanking the bovine growth hormone gene by Southern blot hybridization revealed that they share homology. Screening the bovine genomic library by nick-translated DNA fragment from 5' flanking region leads to conclusion that this sequence is present in 0.1% of clones. Each analysed clone carrying the sequence contains some copies of it.     

The structure of anchorin CII, a collagen binding protein isolated from chondrocyte membrane

cDNA clones for anchorin CII (Mr = 34,000), a collagen-binding protein, were isolated from a lambda gt 11 cDNA library prepared from chick cartilage mRNA. Several overlapping clones were characterized which gave rise to an open reading frame coding for 329 residues and a 3'-untranslated segment of 500 base pairs. The clones were identified as coding for anchorin by hybrid select translation analysis and by comparing the deduced amino acid sequence with the sequence of 10 tryptic peptides of the protein. A hydrophobic domain of 25 residues interrupted with 3 polar residues was identified with the carboxyl-terminal portion. There was no evidence for an aminoterminal signal peptide. Northern analysis revealed that the 5' probe hybridizes to a single 1.7-kilobases (kb) mRNA species, whereas the 3' probe hybridizes to two mRNA species of 1.7 kb and 5 kb, which are present in many cells including chondrocytes, crop cells, and fibroblasts. The level of anchorin mRNA in chick embryo fibroblasts was increased by infection with Rous sarcoma virus.     

Cloned embryonic DNA sequences flanking the mouse immunoglobulin C gamma 3 and C gamma 1 genes

To investigate the DNA surrounding genes for immunoglobulin heavy chain constant (CH) regions, we have isolated two clones bearing a C gamma 3 gene and two bearing a C gamma 1 gene from a library of mouse embryo DNA fragments. The C gamma 3 clones span 8.6 kilobase pairs (kb) on the 5' side of the gene and 6.7 kb on its 3' side, while the C gamma 1 clones together span 13 kb of 5' flanking sequence and 2.5 kb of 3' flanking sequence. Restriction mapping of the C gamma 3 gene indicates that intervening sequences divide the gene into segments of domain size, as in other CH genes. Hybridization of clone fragments to restriction digests of mouse DNA indicates that both the C gamma 1 and C gamma 3 genes probably occur as single copies in the genome. Moreover, the entire cloned sequences on the 5' side of both genes appear to be unique in the genome, indicating that no large common sequences flank CH genes. Restriction data suggest that the C gamma 3 gene is 37-40 kb 5' to the C gamma 1 gene.     

Database for exchangeable gene trap clones: Pathway and gene ontology analysis of exchangeable gene trap clone mouse lines

Gene trapping in embryonic stem (ES) cells is a proven method for large‐scale random insertional mutagenesis in the mouse genome. We have established an exchangeable gene trap system, in which a reporter gene can be exchanged for any other DNA of interest through Cre/mutant lox‐mediated recombination. We isolated trap clones, analyzed trapped genes, and constructed the database for Exchangeable Gene Trap Clones (EGTC) [ http://egtc.jp ]. The number of registered ES cell lines was 1162 on 31 August 2013. We also established 454 mouse lines from trap ES clones and deposited them in the mouse embryo bank at the Center for Animal Resources and Development, Kumamoto University, Japan. The EGTC database is the most extensive academic resource for gene‐trap mouse lines. Because we used a promoter‐trap strategy, all trapped genes were expressed in ES cells. To understand the general characteristics of the trapped genes in the EGTC library, we used Kyoto Encyclopedia of Genes and Genomes (KEGG) for pathway analysis and found that the EGTC ES clones covered a broad range of pathways. We also used Gene Ontology (GO) classification data provided by Mouse Genome Informatics (MGI) to compare the functional distribution of genes in each GO term between trapped genes in the EGTC mouse lines and total genes annotated in MGI. We found the functional distributions for the trapped genes in the EGTC mouse lines and for the RefSeq genes for the whole mouse genome were similar, indicating that the EGTC mouse lines had trapped a wide range of mouse genes.     

Isolation and characterization of expressible cDNA clones for mouse Thy-1: a model system for cDNA expression of cell surface proteins

By using the mouse Thy-1 gene as a model, we have developed a procedure to distinguish functional vs nonfunctional cDNA of lymphocyte surface antigens by transfecting COS-7 monkey cells and testing for expression of cell surface products encoded by the cDNA inserts. By cross-hybridization with a mouse Thy-1 probe, we isolated cDNA clones from a pcD-expression library prepared from mRNA of C5 cells. Two functional clones were distinguished from the remainder by detection of Thy-1.2 on the surface of 0.5% of COS-7 cells transiently transfected by the DEAE-Dextran method. Inclusion of chloroquine in the transfection procedure greatly facilitated the detection of functional cDNA by raising the percentage of expressing cells to 30%. Nucleotide sequencing of one functional cDNA, about 1700 bp long, confirmed that the gene encodes a protein whose sequence agrees with the published Thy-1.2 protein sequence with the additional 31 amino acids attached at the COOH-terminus. A 75 bp 5' untranslated region preceding the coding region contains 50 bp not found in the genomic clones. Comparison indicates that one or more introns are present in the 5' untranslated region, but are not found in the mature mRNA. The first exon may be separated by at least 1 kb intron from the initiation codon. Because the expressible clones are approximately the size of the mRNA seen on Northern blots, we believe that these clones are nearly full-length cDNA. Dilution experiments indicate that this strategy should also be useful for identifying functional cDNA clones for cell surface proteins solely on the basis of their expression in mammalian cells.     

A system for the measurement of gene targeting efficiency in human cell lines using an antibiotic resistance-GFP fusion gene

Gene targeting in a broad range of human somatic cell lines has been hampered by inefficient homologous recombination. To improve this technology and facilitate its widespread application, it is critical to first have a robust and efficient research system for measuring gene targeting efficiency. Here, using a fusion gene consisting of hygromycin B phosphotransferase and 3'-truncated enhanced GFP (HygR-5' EGFP) as a reporter gene, we created a molecular system monitoring the ratio of homologous to random integration (H/R ratio) of targeting vectors into the genome. Cell clones transduced with a reporter vector containing HygR-5' EGFP were efficiently established from two human somatic cell lines. Established HygR-5' EGFP reporter clones retained their capacity to monitor gene targeting efficiency for a longer duration than a conventional reporter system using an unfused 5' EGFP gene. With the HygR-5' EGFP reporter system, we reproduced previous findings of gene targeting frequency being up-regulated by the use of an adeno-associated viral (AAV) backbone, a promoter-trap system, or a longer homology arm in a targeting vector, suggesting that this system accurately monitors H/R ratio. Thus, our HygR-5' EGFP reporter system will assist in the development of an efficient AAV-based gene targeting technology.     

Structure of the gene encoding potato cytosolic pyruvate kinase.

The polymerase chain reaction (PCR) has been used to generate a series of overlapping genomic clones representing 43 bp of 5' untranslated sequence, 63 bp of 3' untranslated sequence and the entire coding sequence of the gene encoding potato cytosolic pyruvate kinase (PKc). This portion of the gene is approximately 4.5 kb in length and is interrupted by three introns, one of which is present in the 5' untranslated region. Southern blot analysis indicates that PKc is encoded by a small gene family, and sequence data from a number of PCR-derived genomic clones indicate that there are as many as six PKc genes. Sequence differences between the PCR-generated genomic clones and a PKc cDNA clone are discussed with respect to the fidelity of Taq polymerase. An alignment of intron placement in the potato PKc gene with intron placement in PK genes from other sources indicates that two of the potato introns correspond to intron positions in other species.     

人淋巴细胞CD2基因5’侧翼序列的克隆和鉴定

本文报告以ＣＤ２ｃＤＮＡ５’端的片段作探针,从人Ｔ淋巴细胞基因组文库筛选阳性重组克隆,经限制性内切酶降解和Ｓｏｕｔｈｅｒｎ杂交分析,证明其中一个阳性克隆的插入片段中含ＣＤ２基因５’侧翼顺序。经插入片段的亚克隆、限制性内切酶图谱及ＤＮＡ序列分析,鉴定出一含转录起始点及其上游序列的４．０ｋｂ片段。将此片段中含转录起始点和两个ＤＮ（ａｓｅ）Ⅰ高敏感位点的２．５ｋｂ片段定向克隆到以虫萤光素酶为报告基因的表达载体ｐＭＧ３中,并用限制性内切酶对此２．５ｋｂ片段作不同程度缺失,构成一系列突变子。这些重组的表达质粒转染人ＪｕｒｋａｔＴ细胞后,以瞬时表达实验分析各突变子驱动虫萤光素酶基因的表达,结果发现在ＣＤ２基因５’上游具有很弱的启动子活性,初步测定该启动子位于－１．２ｋｂ～－９８ｂｐ域。ＣＤ２基因具有弱启动子、强增强子的特点与Ｔ细胞表面其它抗原分子基因是相似的。     

Production of hydrogen peroxide by rabbit articular chondrocytes. Enhancement by cytokines

Recent evidence suggests that reactive oxygen intermediates may play a role in the etiology of cartilage matrix degradation in arthritis. We have previously established that normal articular chondrocytes can functionally act as macrophages. These functions include expression of class II MHC Ag, presentation of Ag and induction of mixed and autologous lymphocyte stimulation. Inasmuch as the production of reactive oxygen intermediates is a hallmark of macrophage activity during inflammatory response, we were interested in examining the ability of normal articular chondrocytes to produce reactive oxygen intermediates. Using the trapped indicator 2',7'-dichlorofluorescin diacetate (DCFH-DA), we measured the levels of intracellular hydrogen peroxide within normal rabbit articular chondrocytes. We found that Concanavalin A induces chondrocytes to rapidly oxidize 2',7'-dichlorofluorescin diacetate to a highly fluorescent dichlorofluorescin in a dose- and time-dependent manner. Fluorescent dichlorofluorescin oxidation by chondrocytes was inhibited by the addition of catalase, an enzyme that detoxifies hydrogen peroxide. Exposure of rabbit chondrocytes to either IFN-gamma or TNF primed the chondrocytes to produce significantly greater amounts of hydrogen peroxide with or without further stimulation. Using scopoletin oxidation as a measure of the release of hydrogen peroxide, we confirmed that chondrocytes released this reactive oxygen intermediate after adherence to serum coated culture plates. Rabbit articular chondrocytes produced and released greater amounts of hydrogen peroxide than pulmonary alveolar macrophages, a well characterized macrophage cell type. These observations suggest that chondrocytes are an important source of reactive oxygen intermediates. Furthermore, the production of reactive oxygen intermediates by chondrocytes may be an important mechanism by which chondrocytes induce structural and functional alterations in cartilage matrix observed during arthritis.