Development of a probe consist of three cosmids to enumerate the chromosome 13 on uncultured lymphocytes or amniocytes using interphase FISH

To produce a reliable probe suitable for aneuploidy detection of chromosome13 on uncultured lymphocytes and amniocytes by fluorescence in situ hybridization [FISH], we used a contig of three overlapping cosmids mapped to 13q12.3. The cosmid DNA carrying the expected sequences of human chromosome 13 was isolated from host cells and labelled with biotin-11-dUTP. The hybridization and detection conditions with FITC-Avidin were optimised using a series of cultured and uncultured lymphocytes and amniocytes. Intensive signals were detected when a combination of three overlapping cosmids was used to enumerate the chromosome 13 on interphase nuclei. An average of 87 and 85.5 percent of interphase cells prepared from lymphocytes and amniocytes showed accurate number of specific signals for chromosome 13. The results obtained in present study indicate that the probe was capable of detecting the copy number of chromosome 13 on interphase cells prepared from peripheral blood or amniotic fluid cells providing that the uncultured amniotic fluid cells are free of cytoplasmic residues, RNA and protein debris

Genetic and physical map of broad host range cosmid pRG930cm

We hereby present the complete sequence and annotation of pRG930cm, a spectinomycin/streptomycin/chloramphenicol-resistant cosmid vector. pRG930cm (17,256 bp; GenBank Accession No.: FM174471) has a broad host range, and is stably maintained by a number of Gram-negative bacteria including Pseudomonas spp, Escherichia coli, Agrobacterium tumefaciens and Azorhizobium caulinodans ORS571. pRG930cm is already widely used and its sequence will aid efficient construction and analysis of cosmid libraries.

Effect of LiCl on Iodine Kinetics in Thyroid Cancer Cell Lines Transduced by Recombinant Adenovirus Containing Sodium Iodide Symporter(NIS) Gene / 대한내분비학회지

BACKGROUND: Lithium is known to increase the retention of iodide in the thyroid gland, or in well differentiated thyroid cancer tissue. The effects of lithium on the function of the sodium iodide symporter (NIS) protein, especially when the lithium is increased in the retention of iodide in NIS-producing cells, the effect of lithium, on the kinetics of undifferentiated thyroid cancer cells transduced by a recombinant adenovirus containing the NIS gene, were checked. METHOD: Human NIS cDNA was inserted into pAxCAwt, a recombinant adenoviral cosmid vector, where the E1 & E2 genes have been deleted, making Rad-hNIS, which was propagated in 293 cells. The iodide uptake was evaluated by the 125I uptake assay in the undifferentiated thyroid cancer cells, ARO, FRO and NPA, following the infection with Rad-hNIS (1 or 10 MOI) in the presence, or absence, of LiCl at optimized concentrations. The iodide efflux was evaluated by the 125I efflux assay, for 1 hour, in the same cells expressing the NIS in the presence, or absence, of LiCl. Similar experiments were performed in the normal thyroid cell line, FRTL-5, cultured in 6H5 media. RESULTS: LiCl, at concentrations over 1.0mM, caused a significant decrease in the cell viability, as evaluated by trypan blue dye exclusion, in a dose dependent manner. When infected with Rad-hNIS, the iodide uptake was not affected by the LiCl in the ARO or NPA cells. However, LiCl(0.1and 1.0mM) increased the iodide uptake by 50 to 100%(vs. control) in the Rad-hNIS transduced FRO cells. In the Rad-hNIS transduced FRO cells, the iodide was released rapidly from the cells, with only 20.7+/-4.8% of the iodide uptake remaining at 1 hour, which was no different in the presence of LiCl (24.5+/-7.9%). The iodide efflux was not affected by the LiCl in the FRTL-5 cells cultured in the presence of TSH. CONCLUSION: These results suggest that the lithium-induced iodide retention in the thyroid gland, or in well differentiated thyroid cancer tissue, is not caused by the effect of the lithium on the NIS function, or the function of proteins or channels, involved in iodide transport via cell membranes. Although the iodide uptake can be markedly increased by the expression of NIS, with the transduction of Rad-hNIS, in undifferentiated thyroid cancer cells, the iodide taken up is rapidly released from the cells. A method for inducing the iodide retention in the cell should be elucidated in order to render the NIS gene therapy effective.

Generation of a series of recombinant herpes simplex viruses which can provide replicating and packaging functions for recombinant adeno-associated virus / 中华实验和临床病毒学杂志

<p><b>BACKGROUND</b>To construct a series of recombinant herpes simplex viruses that can provide replicating and packaging functions for recombinant adeno-associated virus (rAAV), and to select the strains possessing stronger functions for large-scale production of rAAV.</p><p><b>METHODS</b>A set of cosmids that represents the whole genome of HSV-1 was used to generate recombinant HSV-1 expressing rep and cap proteins of AAV-2. An ATG-to-ACG mutation in the start codon of AAV-2 rep protein was generated by site-directed mutagenesis. rep and cap genes, under control of their native promoters, with or without the ATG-to-ACG mutation in the start codon of rep, were inserted into the Xba site of UL2 or UL44 genes, respectively, resulting in the recombinant cosmids cos6-rmc/ UL2, cos56-rc/ UL44 and cos56-rmc/ UL44. Homologous recombination among the HSV-1 fragment on the recombinant cosmids and the rest fragments of HSV-1 genome resulted in three strains of recombinant HSV-1. Together with the one was constructed previously, there were four strains of recombinant HSV-1named HSV1-rc/ UL2, HSV1-rmc/ UL2, HSV1-rc/ UL44 and HSV1-rmc/ UL44 respectively.</p><p><b>RESULTS</b>PCR detection confirmed the existence of rep- gene in the genomes of all four strains of the recombinant HSV-1. Recombinant AAV was produced after infecting the AAV vector cell line that carrying the GFP expression cassette with the four strains of recombinant HSV-1 respectively. However, HSV1-rc/ UL2 and HSV1-rmc/ UL2 produced much more rAAV than HSV1-rc/ UL44 and HSV1c/ UL44 did.</p><p><b>CONCLUSIONS</b>All the four strains of recombinant HSV-1 support rAAV replication and packaging. HSV1 UL2 and HSV1-rmc/ UL2 that provide much stronger functions may be useful for large-scale production of rAAV.</p>

Enhanced Effect of Iodide - Uptake in Thyroid Carcinoma Cells by Infecting Adenovirus - Human Sodium Iodide Symporter (Ad - hNIS) / 대한내분비학회지

BACKGROUND: The sodium-iodide-symporter (NIS) is a plasma membrane glycoprotein with 13 putative transmembrane domains, which is responsible for concentrating iodide into the thyroid by an active transport and provides the mechanism for radioactive-iodine (RAI) therapy for thyroid cancer. However, undifferentiated thyroid cancers and about 2050% of differentiated thyroid cancers do not take up the RAI at therapeutic dose. The NIS has been cloned from rat and human (hNIS) and characterized recently. In an attempt to develop a new therapeutic strategy using hNIS gene for improving the efficacy of RAI therapy in thyroid cancers, we have constructed a recombinant adenovirus encoding the hNIS (Ad-hNIS) and tested its function by an iodide uptake by infecting human thyroid cancer cells. METHODS: RT-PCR was performed to measure an intrinsic hNIS expression in thyroid cancer cell lines, such as NPA, FRO and ARO. To generate the hNIS adenovirus, hNIS cDNA was isolated and ligated into Swa I site of cosmid shuttle vector (pAxCAwt). We have produced recombinant adenovirus by co-transfecting the cosmid with DNA-TPC to 293 cell line. Adenovirus that express (beta-Galactosidase (LacZ) was also prepared by the similar strategy. Adenovirus infection efficiency was measured in three thyroid cancer cell lines. Finally, 24 hours after infection of ad-hNIS into the cells, I125-uptake was measured. RESULTS: Endogenous hNIS expression was detected only in FRO cells but not in NPA, ARO and Hela cells by RT-PCR. X-Gal staining after infection of Ad-LacZ to thyroid cancer cell (NPA, ARO, FRO) showed that an infection rate in ARO cells was 98.5+0.5%, 97.0+0.2% in FRO cells and 75.5+5.0% in NPA cells. We selected ARO cells for the infection of Ad-hNIS due to the highest infection efficiency and the absence of endogenous hNIS expression. When ARO cells were infected with the ad-hNIS, I125 uptake was increased 504+6.4%. CONCLUSION: Overexpression of hNIS gene in thyroid cancer cells elicited over 5 fold increase in I-uptake, suggesting that the Ad-hNIS infection to the thyroid cancer cells may improve the efficiency of radioactive iodine therapy.

Amplification of int-2 in Head and Neck Squamous Cell Carcinomas and Adjacent Mucosa / 대한이비인후과학회지

BACKGROUND AND OBJECTIVES: It is important to identify potential biomarkers of tumorigenesis that can be utilized on histologically normal epithelia to determine the level of risk of tumor development. With the goal of possibly identifying a biomarker for the process of development of head and neck cancer, the amplification of int-2 was observed in patients with head and neck squamous cell carcinoma. MATERIALS AND METHODS: Fluorescence in situ hybridization using cosmid int-2 probe was performed on paraffin-embedded specimens from tumor and tumor-adjacent and tumor-distant epithelia of 20 patients. Buccal mucosa of cancer-free subjects who smoked and did not smoke cigarettes were used as control. Dot blot hybridization using digoxigenin-labeled int-2 probe was also performed on the frozen tissue from tumor and tumor-adjacent epithelia of 14 patients. RESULTS: In in situ hybridization, buccal epithelia of cancer-free subjects who smoked and did not smoke cigarettes, and tumor-distant epithelia of the patients with head and neck squamous cell carcinoma showed no int-2 amplification. However, eleven of tumor tissue (55%) and five of tumor-adjacent epithelia (25%) in 20 cases showed int-2 amplification. In dot blot hybridization, five tumor tissue (35.7%) and 2 tumor-adjacent epithelia (14.3%) in 14 cases, of which tumor tissue were all found to have int-2 amplification, showed int-2 amplification. CONCLUSION: The amplification of int-2 in the tumor tissue and the tumor-adjacent epithelia of the same cases supports the concept of field cancerization or clonal extension. Such genotype parameters may provide a genetic basis for the development of early recurrence or second primary tumors after therapeutic treatment of head and neck squamous cell carcinomas.

Proceedings of the schistosome genome project

"The host-parasite relationship" is a vast and diverse research field which, despite huge human and financial input over many years, remains largely shrouded in mystery. Clearly, the adaptation of parasites to their different host species, and to the different environomental stresses that they represent, depends on interactions with, and responses to, various molecules of host and/or parasite origin. The schistosome genome project is a primary strategy to reach the goal; this systematic research project has successfully developed novel technologies for qualitative and quantitative characterization of schistosome genes and genome organization by extensive international collaboration between top quality laboratories. Schistosomes are a family of parasitic blood flukes (Phylum Platyhelminthes), which have seven pairs of autosomal chromosomes and one pair of sex chromosomes (ZZ for a male worm an ZW for a female), of a haploid genome size of 2.7X10 8 base pairs (Simpson et al. 1982). Schistosomes are ideal model organisms for the development of genome mapping strategies since they have a small genome size comparable to that of well-characterized model organisms such as Caenorhabditis elegans (100 Mb) and Drosophila (165Mb), and contain functional genes with a high level of homology to the host mammalian genes. Here we summarize the current progress in the schistosome genome project, the information of 3.047 transcribed genes (Expresses Sequence Tags; EST), complete sets of cDNA and genomic DNA libraries (including YAC and cosmid libraries) with a mapping technique to the well defined schistosome chromosomes. The schistosome genome project will further identify and charaterize the key molecules that are responsable for host-parasite adaptation, i.e., successful growth developement, maturation and reproduction of the parasite within its host in the near future.

Cloning of the Form I-antigen Genes from Shigella sonnei KNIH104

Shigella sonnei KNIH104S, which was selected by Korean National Institute of Health, expresses form I-antigen as a somatic antigen. In this study, we cloned the genes responsible for form I-antigen synthesis from S. sonnei KNIH104S. A Sau3AI-generated cosmid library of S. sonnei KNIH104S plasmids were transfected into E. coli LE392 and transfectants were tested for agglutination with antiserum against S. sonnei form I-antigen. A clone, JH222, showing the strongest agglutination activity was chosen for further analysis. A recombinant cosmid, pJH222, was isolated from the strain JH222 and retransfected into E. coli LE392. All of the transfectants agglutinated with antiserum against form I-antigen, indicating that pJH222 carried the genes required for S. sonnei form I-antigen synthesis. Restriction analysis of pJH222 revealed a 38 kb insert, which was confirmed by Southern hybridization analysis to be present on a large plasmid of S. sonnei KNIH104S.

Isolation of virulence genes directing GPI synthesis by functional complementation of Leishmania

Recent advances in molecular genetics of Leishmania parasites prompted us to develop methods of functional genetic complementation in Leishmania and apply them to the isolation of genes involved in the biosynthesis of the virulence determinant LPG, an abundant GPI-anchored polysaccharide. LPG1, the gene product identified by complementation of our R2D2 LPG- mutant, may be a glycosyltransferase responsible for the addition of galactofuranose to the nascent chain. As galactofuranose is not found in mammalian cells, inhibition of the addition of this sugar could be exploited for chemotherapy. Overall, the success of the functional complementation approach opens the way to the identification of a variety of genes involved in pathogenesis and parasitism