Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.4.2.8 (hypoxanthine-guanine phosphoribosyltransferase)
 2,527 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We developed a one-step real-time duplex reverse transcription PCR (RT-PCR) method using the LightCycler platform. This method allows simultaneous reverse transcription and real-time PCR amplification of two mRNAs of specific genes of interest (analyte genes) and mRNA of constantly transcribed genes (housekeeping genes) in a single-tube reaction. Specimen total nucleic acids were used because eukaryotic cDNA is discriminated from genomic DNA using exon-spanning primers and/or fluorescence resonance energy transfer (FRET) probes. Transcripts of murine arginase I and hypoxanthine-phosphoribosyl transferase (HPRT; housekeeping gene) or murine arginase II analyte and porphobilinogen deaminase (PBGD; housekeeping gene) were quantified in such duplex RT-PCRs. Twenty-minute reverse transcription reactions at 55 degrees C followed by 18 high-stringency step-down thermal cycles and 25 relaxed-stringency fluorescence acquisition cycles produced sensitive and accurate RT-PCR results. Fluorescent signal spillover between channels was fully compensated. A matrix of duplex PCRs at variable ratios of target standards yielded equations for factors that correct PCR-specific target ratio-dependent deviations in quantification. The one-step real-time duplex RT-PCRs reliably and accurately determined 10-10,000 copies of each target over a 100,000-fold range of target copy ratios (analyte to housekeeping mRNA = 10(-2.5)-10(2.5)) in a single assay.
 ...
PMID:One-step real-time duplex reverse transcription PCRs simultaneously quantify analyte and housekeeping gene mRNAs. 1503 67

Urea cycle disorders are incurable enzymopathies that affect nitrogen metabolism and typically lead to hyperammonemia. Arginase deficiency results from a mutation in Arg1, the enzyme regulating the final step of ureagenesis and typically results in developmental disabilities, seizures, spastic diplegia, and sometimes death. Current medical treatments for urea cycle disorders are only marginally effective, and for proximal disorders, liver transplantation is effective but limited by graft availability. Advances in human induced pluripotent stem cell research has allowed for the genetic modification of stem cells for potential cellular replacement therapies. In this study, we demonstrate a universally-applicable CRISPR/Cas9-based strategy utilizing exon 1 of the hypoxanthine-guanine phosphoribosyltransferase locus to genetically modify and restore arginase activity, and thus ureagenesis, in genetically distinct patient-specific human induced pluripotent stem cells and hepatocyte-like derivatives. Successful strategies restoring gene function in patient-specific human induced pluripotent stem cells may advance applications of genetically modified cell therapy to treat urea cycle and other inborn errors of metabolism.
 ...
PMID:Restoring Ureagenesis in Hepatocytes by CRISPR/Cas9-mediated Genomic Addition to Arginase-deficient Induced Pluripotent Stem Cells. 2789 91