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Oi Lian Kon

Publications and source records attributed to Oi Lian Kon.

9 recordsLinked to original sources
PubMed2006

An isothermal method for whole genome amplification of fresh and degraded DNA for comparative genomic hybridization, genotyping and mutation detection.

Molecular genotyping has important biomedical and forensic applications. However, limiting amounts of human biological material often yield genomic DNA (gDNA) in insufficient quantity and of poor quality for a reliable analysis. This motivated the development of an efficient whole genome amplification method with quantitatively unbiased representation usable on fresh and degraded gDNA. Amplification of fresh frozen, formalin-fixed paraffin-embedded (FFPE) and DNase-degraded DNA using degenerate oligonucleotide-primed PCR or primer extension amplification using a short primer sequence bioinformatically optimized for coverage of the human genome was compared with amplification using current primers by chromosome-based and BAC-array comparative genomic hybridization (CGH), genotyping at short tandem repeats (STRs) and single base mutation detection. Compared with current primers, genome amplification using the bioinformatically optimized primer was significantly less biased on CGH in self-self hybridizations, and replicated tumour genome copy number aberrations, even from FFPE tissue. STR genotyping could be performed on degraded gDNA amplified using our technique but failed with multiple displacement amplification. Of the 18 different single base mutations 16 (89.5%) were correctly identified by sequencing gDNA amplified from clinical samples using our technique. This simple and efficient isothermal method should be helpful for genetic research and clinical and forensic applications.

Base Sequence↗
PubMed2006

FAT10 plays a role in the regulation of chromosomal stability.

Aneuploidy is a key process in tumorigenesis. Dysfunction of the mitotic spindle checkpoint proteins has been implicated as a cause of aneuploidy in cells. We have previously reported that FAT10, a member of the ubiquitin-like modifier family of proteins, is overexpressed in several gastrointestinal and gynecological cancers. Here we show that FAT10 interacts with MAD2, a spindle checkpoint protein, during mitosis. Notably, we show that localization of MAD2 at the kinetochore during the prometaphase stage of the cell cycle was greatly reduced in FAT10-overexpressing cells. Furthermore, compared with parental HCT116 cells, fewer mitotic cells were observed after double thymidine-synchronized FAT10-overexpressing cells were released into nocodazole for more than 4 h. Nonetheless, when these double thymidine-treated cells were released into media, a similar number of G1 parental and FAT10-overexpressing HCT116 cells was observed throughout the 10-h time course. Additionally, more nocodazole-treated FAT10-overexpressing cells escape mitotic controls and are multinucleate compared with parental cells. Significantly, we observed a higher degree of variability in chromosome number in cells overexpressing FAT10. Hence, our data suggest that high levels of FAT10 protein in cells lead to increased mitotic nondisjunction and chromosome instability, and this effect is mediated by an abbreviated mitotic phase and the reduction in the kinetochore localization of MAD2 during the prometaphase stage of the cell cycle.

Aneuploidy↗
PubMed2006

In vivo liver electroporation: optimization and demonstration of therapeutic efficacy.

Adverse effects (death and leukemogenesis) from viral vector-mediated gene therapy have renewed interest in plasmids as safer, more scalable, simple, and cost-effective vectors. Electroporation and hydrodynamic delivery are two techniques that improve the efficiency of plasmid-mediated gene transfer. The liver is a good tissue platform for targeted transfer of therapeutically relevant genes for correction of metabolic disorders, for example, hemophilia A. However, in vivo electroporation of liver has not yet been shown to achieve therapeutic efficacy of systemically active, secreted transgenic proteins. We have investigated the effect of field strength, pulse duration, pulse number, electrical waveforms, electrode contact area, plasmid administration routes, and injection technique on the efficiency of in vivo electrotransfer of naked plasmid to liver. Plasmid injection into a systemic vein was superior to intrahepatic injection. Unlike in vivo muscle electroporation, high-voltage pulses and microsecond pulses offered no advantage. Optimal electroporation conditions were 8-10 uni- or bipolar pulses of 20 msec, each at 250 V/cm. Using a nonhydrodynamic technique that greatly enhanced electrotransfer efficiency with minimal tissue injury, we demonstrate for the first time that liverdirected in vivo electroporation of factor VIII cDNA achieved significant phenotypic correction in hemophilic mice.

Animals↗
PubMed2006

Genetic engineering for haemophilia A.

At first sight, haemophilia A would appear to be an ideal candidate for treatment by gene therapy. There is a single gene defect; cells in different parts of the body, but especially the liver, produce Factor VIII, and only 5% of normal levels of Factor VIII are necessary to prevent the serious symptoms of bleeding. This review attempts to outline the status of gene therapy at present and efforts that have been made to overcome the difficulties and remaining problems that require solving. Undoubtedly, success will be achieved, but it is likely that considerably more work will be necessary before experimental models can be introduced into the clinic with any likelihood of success. The most successful results in animals that may have clinical application were from introducing the Factor VIII gene to newborn animals before antibodies are produced, presumably inducing a state of tolerance.

Animals↗
PubMed2005

Wavelet transformations of tumor expression profiles reveals a pervasive genome-wide imprinting of aneuploidy on the cancer transcriptome.

Aneuploidy is frequently observed in many human cancers, but its global effects on the cancer transcriptome are controversial. We did a systematic and unbiased genome-wide survey to determine the extent a tumor's abnormal karyotype (chromosomal amplifications and deletions) is detectably "imprinted" onto that tumor's gene expression profile. By using a novel methodology employing wavelet transform signal-processing algorithms to identify genomic regions of coordinated gene expression (wavelet variance scanning), we analyzed a series of gastric cancer cell lines and identified >100 genomic regions exhibiting distinct patterns of subtle but significant coordinated transcription, ranging from tens to hundreds of genes. A large majority (80%) of these regions could be specifically localized to a site of detectable genomic amplification or deletion; reciprocally, up to 47% of the total aneuploidy in each of the individual cell lines could be directly inferred from the gene expression data. Genome-wide portraits of tumor aneuploidy can thus be successfully reconstructed solely from gene expression data, implying that the effects of aneuploidy must be pervasively and globally imprinted within the cancer transcriptome. Aneuploidy may contribute to tumor behavior not just by affecting the expression of a few key oncogenes and tumor suppressor genes but also by subtly altering the expression levels of hundreds of genes in the oncogenome.

Aneuploidy↗
PubMed2005

FVIII gene delivery by muscle electroporation corrects murine hemophilia A.

BACKGROUND: Hemophilia A treatment relies on costly factor VIII (FVIII) replacement that may transmit iatrogenic viral diseases. Viral vectors and cell implants are being developed as improvements. We investigated in vivo electroporation of naked DNA as a safe and simple method for correcting FVIII deficiency. METHODS: B-domain-deleted murine FVIII cDNA expression plasmids were constructed with CMV and elongation factor 1alpha promoters for characterisation in murine C2C12 myoblasts. The construct conferring highest in vitro FVIII secretion was electroporated into skeletal muscle of FVII null mice in vivo for phenotypic correction using a protocol that minimised tissue injury. RESULTS: B-domain-deleted murine FVIII cDNA plasmids induced FVIII secretion from stably transfected C2C12 myoblasts (0.54+/-0.20 mU/day/10(5) cells). Phenotypic correction of hemophilic mice was more consistently achieved using a protocol for in vivo electroporation of gastrocnemius muscle with FVIII cDNA that reduced tissue injury by the use of plate electrodes, hyaluronidase pre-treatment and lower field strength. This technique was associated with <10% muscle necrosis. Activated partial thromboplastin time decreased from 51.4+/-3.3 to 34.7+/-1.1 (mean+/-s.e.m.) seconds (p=0.0004) following in vivo electroporation (0.1 mg plasmid/limb; 8x20 ms pulses, 175 V/cm, 1 Hz) of hemophilic mice. All hemophilic mice (8/8) survived hemostatic challenge after muscle electroporation with FVIII cDNA, whereas all (9/9) untreated hemophilic mice died. Plasmid DNA was detectable only in electroporated muscle and not in all other organs tested, including gonads. CONCLUSION: In vivo intramuscular electroporation of naked FVIII plasmid successfully corrects murine hemophilia.

Animals↗
PubMed2003

A combined comparative genomic hybridization and expression microarray analysis of gastric cancer reveals novel molecular subtypes.

Comparative genomic hybridization (CGH), microsatellite instability (MSI) assays, and expression microarrays were used to molecularly subclassify a common set of gastric tumor samples. We identified a number of novel genomic aberrations associated with gastric cancer and discovered that gastric tumors could be grouped by their expression profiles into three broad classes: "tumorigenic," "reactive," and "gastric-like." Patients with gastric-like tumors exhibited a significantly better overall survival than patients belonging to the other two classes (P < 0.05). A novel supervised learning methodology for multiclass prediction was used to identify optimal predictor gene sets that accurately predicted the class of an unknown tumor sample. These predictor sets may prove useful in the development of new diagnostic applications for gastric cancer staging and prognostication.

Adenocarcinoma↗
PubMedDate not supplied

Human liver-derived cells stably modified for regulated proinsulin secretion function as bioimplants in vivo.

BACKGROUND: Insulin deficiency is currently treated with pharmacological insulin secretagogues, insulin injections or islet transplants. Secondary failure of pharmacological agents is common; insulin injections often fail to achieve euglycemic control; and islet transplants are rare. Non-beta cells capable of regulated insulin secretion in vivo could be a functional cure for diabetes. Hepatocytes are good candidates, being naturally glucose-responsive, protein-secreting cells, while the liver is positioned to receive direct nutrient signals that regulate insulin production. METHODS: Human liver-derived Chang cells were modified with a plasmid construct in which a bifunctional promoter comprising carbohydrate response elements and the human metallothionein IIA promoter controlled human proinsulin cDNA expression. Secretory responses of stable cell clones were characterized in vitro and in vivo by proinsulin radioimmunoassay. RESULTS: Transfected Chang cells secreted 5-8 pmol proinsulin/10(6) cells per 24 h in continuous passage for at least a year in response to 5-25 mM glucose and 10-90 microM zinc in vitro. Glucose and zinc synergistically increased proinsulin production by up to 30-fold. Non-glucose secretagogues were also active. Glucose transporter 2 (GLUT2) and glucokinase cDNA co-transfection enhanced glucose responsiveness. Intraperitoneally implanted Chang cells secreted proinsulin in scid and Balb/c mice. Serum proinsulin levels were further increased 1.3-fold (p<0.05) after glucose and 1.4- to 1.6-fold (p<0.005) after zinc administration in vivo. CONCLUSIONS: These results are the first to demonstrate stable proinsulin production in a human liver-derived cell line with activity in vitro and in vivo and provide a basis for engineering hepatocytes as in vivo bioimplants for future diabetes treatment.

Animals↗