Hypoxanthine-guanine Phosphoribosyltransferase 1 Research Articles

Hypoxanthine guanine phosphoribosyltransferase 1, a target of miR-125b-5p, promotes cell proliferation and invasion in head and neck squamous cell carcinoma

The mechanism of hypoxanthine-guanine phosphoribosyltransferase 1 (HPRT1) upregulation and its function in head and neck squamous cell carcinoma (HNSCC) remains obscure. Herein, the expression and function of HPRT1 and the mechanism underlying its upregulation in HNSCC were explored. Firstly, the expression of HPRT1 and its prognostic values were simultaneously validated using bioinformatic analysis and quantitative real-time PCR (qRT-PCR), and immunohistochemistry staining with local HNSCC samples. The effects of HPRT1 knockdown on proliferation and invasion of HNSCC cells were detected using cell counting kit-8 (CCK-8), plate clone formation, Transwell invasion, nude mouse xenograft model assays. Moreover, the miRNA targeting HPRT1 was validated using dual-luciferase report assay, qRT-PCR and Western blot analysis. The functions of miRNA targeting HPRT1 and its dependence on HPRT1 were further investigated in HNSCC. The results indicated that HPRT1 was highly expressed in HNSCC tissues and cells, which positively correlated with advanced tumor progression and predicted poor prognosis in patients with HNSCC. HPRT1 knockdown markedly inhibited proliferation and invasion of HNSCC cells both in vitro and in vivo. MiR-125b-5p, which was downregulated and positively correlated with a favorable outcome for patients, directly targeted and downregulated HPRT1 expression, and subsequently suppressed cell proliferation and invasion in HNSCC. Collectively, the present study demonstrates that HPRT1 upregulation, at least partially caused by miR-125b-5p downregulation, could promote the malignant progression of HNSCC, highlighting the potential application of the miR-125b-5p/HPRT1 axis as a novel indicator and target in the diagnosis and treatment of HNSCC.

Human Papillomavirus E7 and p16INK4a mRNA Multiplexed Quantification by a QuantiGeneTM Proof-of-Concept Assay Sensitively Detects Infection and Cervical Dysplasia Severity.

Persistent infection with human papillomavirus (HPV) can lead to cervical cancer (CxCa). During the progression to CxCa, the expression of HPV oncogenes E6 and E7 is upregulated. In turn, cellular proteins such as p16INK4a are also modulated. The combined detection of HPV oncogenes and cellular biomarkers indicative for dysplasia could be informative and convey better specificity than the current HPV tests that cannot discriminate transient infection from dysplastic changes. The QuantiGeneTM 2.0 Plex Assay platform was chosen for the effective multiplexing and quantitative detection of seven HPV-E7 mRNA targets (HPV6, 16, 18, 31, 45, 59, and 68) and the cellular mRNA of p16INK4a as a biomarker for HPV-induced transformation. Actin-beta (ACTB) and hypoxanthine-guanine phosphoribosyltransferase 1 (HPRT1) were included as reference markers. Sequences for the specific capture and detector probes were customized and developed by ThermoFisher and formulated as a QuantiGene proof-of-concept (QG-POC) plex-set. The crude lysates of the HPV-positive cervical cancer cell lines CaSki (HPV16), HeLa (HPV18), MRHI-215 (HPV45), Erin59 (HPV59), ME180 (HPV68), and the HPV-negative cell line C33A, as well as liquid-based cytology smear samples (n = 441) were analyzed. The study was a proof-of-concept evaluating the feasibility of the platform. Logistic regression and receiver operating characteristic (ROC) analyses were performed to test for the sensitivity and specificity of HPV detection and dysplastic stage discrimination. A QG-POC assay specifically and sensitively detects the HPV-E7 mRNA of seven different genotypes with an assay linearity between 20 and 13,000 cells. Cellular mRNA was detected from the crude lysates of cell lines and of cellular material from clinical liquid-based cytology smear samples. By combining HPV-E7 and p16INK4a expression normalized to ACTB, high-grade dysplasia (HCIN) and invasive cervical cancer (CxCa) were detectable, discriminable, and correlated to the biomarker expression strength. The ROC analysis from the multivariate logistic regression model including HPV-E7 and p16 INK4a resulted in an AUC of 0.74, at the optimal cut-off (sensitivity: 70.4%; specificity: 66.0%) for HCIN detection. CxCa was detected with an AUC of 0.77 (sensitivity: 81.8%, specificity: 77.4%). The QG-POC assay is sufficiently sensitive to detect and quantify HPV-E7 and cellular mRNA species. Multiplexing allows the specific detection of at least 10 analytes in a single reaction. Determining the abundance of E7 and p16INK4a transcripts when normalized to ACTB is informative about the presence of cervical dysplasia and potentially discriminates between low-grade and high-grade dysplasia and invasive cervical cancer. Further studies including more HPV genotypes and biomarkers are warranted.

Therapeutic gene correction for Lesch-Nyhan syndrome using CRISPR-mediated base and prime editing

Lesch-Nyhan syndrome (LNS) is inherited as an X-linked recessive genetic disorder caused by mutations in hypoxanthine-guanine phosphoribosyl transferase 1 (HPRT1). Patients with LNS show various clinical phenotypes, including hyperuricemia, gout, devastating behavioral abnormality, intellectual disability, and self-harm. Although uric acid overproduction can be modulated with the xanthine oxidase inhibitor allopurinol, there exists no treatment for behavioral and neurological manifestations of LNS. In the current study, CRISPR-mediated base editors (BEs) and prime editors (PEs) were utilized to generate LNS-associated disease models and correct the disease models for therapeutic approach. Cytosine BEs (CBEs) were used to induce c.430C>T and c.508C>T mutations in HAP1 cells, and then adenine BEs (ABEs) were used to correct thesemutations without DNA cleavage. PEs induced a c.333_334ins(A) mutation, identified in a Korean patient with LNS, in HAP1 cells, which was corrected in turn by PEs. Furthermore, improved PEs corrected the same mutation in LNS patient-derived fibroblasts by up to 14% without any unwanted mutations. These results suggest that CRISPR-mediated BEs and PEs would be suggested as a potential therapeutic strategy of this extremely rare, devastating genetic disease.

The Correlation Between Children’s Acute Lymphoblastic Leukemia Drug Resistance System Induced by Metabolomics-Based 6-Mercaptopurine and Hypoxanthine-Guanine Phosphoribosyl Transferase 1 Protein

This study aimed to explore 6-mercaptopurine (MP)-induced children’s acute lymphoblastic leukemia (ALL) drug resistance system and leukemia hypoxanthine-guanine phosphoribosyl transferase 1 (HPRT1) protein. Based on metabonomics, drug resistance of 6MP-Reh cell line was established by increasing concentration administration method, and the degree of drug resistance of 6MP-Reh was verified by apoptosis test, western blotting (WB) test, and drug sensitivity test. The changes of tissue inhibitor of matrix metalloproteinase (TIMP) and thioguanosine monophosphate (TGMP) in drug-resistant cells were detected through liquid chromatograph (LC)/mass spectrometer (MS). The 6MP-Reh-wt cell line was established by lentivirus infection, so as to verify the correlation between HPRT1 and drug resistance mechanism. The results showed that the inhibition concentration (IC50) value, cell vitality (CV), apoptosis rate, and 6-MP content of 6MP-Reh were higher hugely than those of Reh (P < 0.05). The contents of HPRT1, TIMP, and TGMP in 6MP-Reh cells were lower sharply than the contents of Reh cells (P < 0.001). The IC50 value of 6MP-Reh-wt was also lower steeply than the value of 6MP-Reh (P < 0.001), and the concentrations of TIMP and TGMP increased obviously (P < 0.05). Therefore, it indicated that the mutation of HPRT1 in drugresistant cell lines could lead to a decrease in their viability and cause leukemia cells to develop resistance to 6-MP. In addition, HPRT1 gene could improve their resistance to 6-MP.

Screening of reference genes in tiger puffer (Takifugu rubripes) across tissues and under different nutritional conditions.

The present study was aimed at screening suitable reference genes for quantitative real-time polymerase chain reaction (qRT-PCR) in tiger puffer (Takifugu rubripes), an important aquaculture species in Asia and also a good model species for lipid research. Specifically, this reference gene screening was targeted at standardization of gene expression in different tissues (liver, muscle, brain, intestine, heart, eye, skin, and spleen) or under different nutritional conditions (starvation and different dietary lipid levels). Eight candidate reference genes (ribosomal protein L19 and L13 (RPL19 and RPL13), elongation factor-1 alpha (EF1α), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), hypoxanthine guanine phosphoribosyl transferase1 (HPRT1), beta-2-Microglobulin (B2M), 18S ribosomal RNA (18SrRNA), and beta actin (ACTB)) were evaluated with four algorithms (geNorm, NormFinder, BestKeeper, and comparative ΔCt method). The results showed that different algorithms generated inconsistent results. Based on these findings, RPL19, EF1α, 18SrRNA, and RPL13 were relatively stable in different tissues of tiger puffer. During starvation conditions, ACTB/RPL19 was the best reference gene combination. Under different dietary lipid levels, ACTB/RPL13 was the most suitable reference gene combination. The present results will help researchers to obtain more accurate results in future qRT-PCR analysis in tiger puffer.

SUN-462 Lesch-Nyhan syndrome: A Case Report

Lesch-Nyhan syndrome, a rare inborn error of metabolism, characterized by Hyperuricemia, Spectrum of neurological dysfunctions, and Cognitive and behavioral disturbances, is a rare anomaly caused by a deficiency of the enzyme hypoxanthine-guanine phosphoribosyl transferase-1 (HGPRT-1), produced by mutations in the HPRT gene located on the X-chromosom leading to the overproduction of purine and accumulation of uric acid. There is no medical treatment to alleviate the symptoms of self-mutilation except dental intervention. The disease affects mainly male child, and females are asymptomatic carriers. We report a case of a 13 year old male child from Ethiopia, Who presented with Worsening of self-injurious behavior and failure to pass urine of 03 days duration. Initially presented with rigidity of the extremities at the age of 4 months and later neck rigidity with intermittent arching of the back. Family noticed passage of whitish grit like material in urine. He had poor appetite, irritability and poor weight gain. For the above complains he was diagnosed to have CP and advised to have physiotherapy. For lack of improvement, family discontinued follow-up. At the age of 3 years, he started to experience a self-injurious behavior by biting initially his lips and subsequently his hands and upper arms with ulceration of the lips and loss of finger nails and later developed head banging and rubbing of the extremities against the floor and nearby object. He developed aggression to his family with biting and pinching. Subsequently started on Clonazepam and Trihexyphenidyl with the diagnosis of dystonic CP, which showed slight improvement. Resperidone was started for the self-injurious behavior, On current presentation, he had poor feeding, low grade intermittent fever, worsening of passage of whitish stone via urine and worsening of self-injurious behavior. He has had recurrent chest and urinary tract infections. His mom and uncle have knee pain and she is taking Allopurinol. There is no history consanguinity in the family. The delivery was in hospital at term, and had uneventful perinatal history. He is the 2nd child to the family, fully vaccinated with adequate nutritional history. Brothers are 17 and 7 years old, grade 10 and 3 students, both apparently healthy. On examination, he is chronically sick looking, irritable. He is severely wasted, stunted and BMI was 17.1. He has ulcerated lips with tissue loss, ulcerated tongue (anterior 2/3 tongue and ulcerated and sloughed nail beds. On CNS he is conscious with intact Cranial nerves; Motor shows wasted muscle bulk but comparable on both sides. Power was 3/5 and had increased tone in all extremities. The DTR was 2/4 in ankle and knee. Plantar reflex was equivocal and there was no clonus or meningeal signs. Sensation to pain and touch was intact. He was admitted and bilateral percutaneous nephrostomy was inserted to relieve ureteric obstruction and medically managed for UTI, multiple electrolyte abnormality due to GI and urinary loss. The AKI resolved with standing ESKD due to obstructive uropathy following nephrolithiasis. In pediatric patients, self-mutilation is seen in genetic syndromes like LNS, Cornelia de Lange, Gilles de la Tourette syndrome, mental retardation, encephalitis, congenital malformation. A careful evaluation is, essential toward making the appropriate diagnosis of LNS.

Selection of suitable reference genes for gene expression studies in myxosporean (Myxozoa, Cnidaria) parasites

Myxozoans (Cnidaria: Myxozoa) are an extremely diversified group of endoparasites some of which are causative agents of serious diseases in fish. New methods involving gene expression studies have emerged over the last years to better understand and control myxozoan diseases. Quantitative RT-PCR is the most extensively used approach for gene expression studies. However, the accuracy of the results depends on the normalization of the data to reference genes. We studied the expression of eight commonly used reference genes, adenosylhomocysteinase (AHC1), beta actin (ACTB), eukaryotic translation elongation factor 2 (EF2), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), hypoxanthine-guanine phosphoribosyltransferase 1 (HPRT1), DNA-directed RNA polymerase II (RPB2), 18S ribosomal RNA (18S), 28S ribosomal RNA (28S) across different developmental stages of three myxozoan species, Sphaerospora molnari, Myxobolus cerebralis and Ceratonova shasta, representing the three major myxozoan linages from the largest class Myxosporea. The stable reference genes were identified using four algorithms: geNorm, NormFinder, Bestkeeper and ΔCq method. Additionally, we analyzed transcriptomic data from S. molnari proliferative and spore-forming stages to compare the relative amount of expressed transcripts with the most stable reference genes suggested by RT-qPCR. Our results revealed that GAPDH and EF2 are the most uniformly expressed genes across the different developmental stages of the studied myxozoan species.

Generation of cell strains containing point mutations in HPRT1 by CRISPR/Cas9

Mutations in Hypoxanthine-guanine Phosphoribosyltransferase1 (HPRT1) gene can lead to metabolic disorder of hypoxanthine and guanine metabolism, and other severe symptoms such as hypophrenia, gout, and kidney stones, called the Lesch-Nyhan disease (LND). Although the mutations are widely distributed throughout the HPRT1 gene, there are some isolated hot spots. In this study, we aim to introduce two previously reported hot spots, c.508 C>T and c.151 C>T, which could lead to premature translational termination in HPRT1 gene. Through CRISPR/Cas9 mediated homology-directed repair (HDR) by using single-stranded oligo-deoxyribonucleotides (ssODN) as donor template, we obtained cell clones containing these two mutations in HEK293T or HeLa cells. Targeted mutation of c.508 C>T and c.151 C>T reached to 16.3% and 10%, respectively. We further detect HPRT1 protein levels with Western blot and enzyme activity with 6-TG in 5 different cell clones. HPRT1 protein and its enzymatic activity both was hardly detected in homozygous mutant cells, while reduced HPRT1 protein expression and enzymatic activity was detected in heterozygous mutant cells. Our study will be beneficial to those who working on generation of cell or animal models of HRPT1 mutations, and provides a basis for further investigations on the genetic mechanism of Lesch-Nyhan disease.

Understanding the structure-function relationship of HPRT1 missense mutations in association with Lesch–Nyhan disease and HPRT1-related gout by in silico mutational analysis

The nucleotide salvage pathway is used to recycle degraded nucleotides (purines and pyrimidines); one of the enzymes that helps to recycle purines is hypoxanthine guanine phosphoribosyl transferase 1 (HGPRT1). Therefore, defects in this enzyme lead to the accumulation of DNA and nucleotide lesions and hence replication errors and genetic disorders. Missense mutations in hypoxanthine phosphoribosyl transferase 1 (HPRT1) are associated with deficiencies such as Lesch–Nyhan disease and chronic gout, which have manifestations such as arthritis, neurodegeneration, and cognitive disorders. In the present study, we collected 88 non-synonymous single nucleotide polymorphisms (nsSNPs) from the UniProt, dbSNP, ExAC, and ClinVar databases. We used a series of sequence-based and structure-based in silico tools to prioritize and characterize the most pathogenic and stabilizing or destabilizing nsSNPs. Moreover, to obtain the structural impact of the pathogenic mutations, we mapped the mutations to the crystal structure of the HPRT protein. We further subjected these mutant proteins to a 50 ns molecular dynamics simulation (MDS). The MDS trajectory showed that all mutant proteins altered the structural conformation and dynamic behavior of the HPRT protein and corroborated its association with LND and gout. This study provides essential information regarding the use of HPRT protein mutants as potential targets for therapeutic development.

Evaluation of candidate reference genes for investigation of the uterine tissue and corpus luteum of pigs on day 6 after artificial insemination

Housekeeping genes (HKG) are genes necessary for the maintenance of basal cellular functions, regardless of the specific roles within a tissue. It, therefore, is expected that these genes will maintain a relatively constant expression profile when there are varying physiological conditions. The identification of tissue specific reference genes is highly important for the normalization of gene expression profiles among different tissues. In this sow study, the objective was to identify stable reference genes in the uterine tissue and corpus luteum (CL), 6 days post-artificial insemination. The stability of ubiquitin (UBB), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), DNA topoisomerase 2-beta (TOP2B), histone H3 (H3F3A) and hypoxanthine-guanine phosphoribosyltransferase 1 (HPRT1) abundances of mRNA were evaluated using the Bestkeeper technique. Briefly, total RNA was extracted for each tissue from 20 gilts (n = 20), processed by RT-qPCR and submitted to analysis using the Bestkeeper technique, which allowed for the evaluation of the consistency in abundance of mRNA for the reference genes. For all evaluated genes, the abundance of mRNA was relatively consistent in the uterine tissue, with the greatest abundance being for the GAPDH and TOP2B genes. The analysis of these genes in the CL, however, indicated there was a relatively greater variation of mRNA abundance for the various reference genes. Data suggest that UBB was the reference gene with the most consistent relative abundance of mRNA and that this gene could be used as a reference for corpora lutea analyses of mRNA.

Absence of Beta-2 microgloblulin (B2M) and hypoxanthine-guanine phosphoribosyl transferase-1(HPRT1) gene modulation in U87MG and U251 Glioblastoma cell lines subjected to cobalt chloride mediated hypoxia

Hypoxia induces broad variations in expression levels of various housekeeping genes. Random selection of housekeeping genes as endogenous control in real time PCR based studies in hypoxia may lead to erroneous interpretation of results and hence such studies require careful selection of valid endogenous controls. In this context, we analyzed the suitability of four most commonly used housekeeping genes as endogenous controls in glioblastoma cell lines exposed to hypoxic conditions. Glioblastoma cell lines U87MG and U251 were subjected to normoxia and cobalt chloride induced hypoxia over varying dosages and time. Expression profiles of four housekeeping genes namely β-2 microglobulin (B2M), hypoxanthine-guanine phosphoribosyl transferase-1 (HPRT1), β-actin (ACTB) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were analyzed by RT-qPCR. The results were statistically analyzed to select reference genes with stable expression profiles. The statistically analyzed results were validated by using NormFinder and BestKeeper softwares. Through this work we report that B2M and HPRT1 exhibit stable expression profile under hypoxic and normoxic experimental conditions. On the other hand, expression levels of GAPDH and ACTB were found to be significantly regulated by hypoxia. Thus B2M and HPRT1 should be considered as candidate endogenous controls for studies with similar experimental setting.

Forward genetic screen of human transposase genomic rearrangements.

BackgroundNumerous human genes encode potentially active DNA transposases or recombinases, but our understanding of their functions remains limited due to shortage of methods to profile their activities on endogenous genomic substrates.ResultsTo enable functional analysis of human transposase-derived genes, we combined forward chemical genetic hypoxanthine-guanine phosphoribosyltransferase 1 (HPRT1) screening with massively parallel paired-end DNA sequencing and structural variant genome assembly and analysis. Here, we report the HPRT1 mutational spectrum induced by the human transposase PGBD5, including PGBD5-specific signal sequences (PSS) that serve as potential genomic rearrangement substrates.ConclusionsThe discovered PSS motifs and high-throughput forward chemical genomic screening approach should prove useful for the elucidation of endogenous genome remodeling activities of PGBD5 and other domesticated human DNA transposases and recombinases.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-2877-x) contains supplementary material, which is available to authorized users.

Data supporting the design and evaluation of a universal primer pair for pseudogene-free amplification of HPRT1 in real-time PCR.

Hypoxanthine-guanine phosphoribosyltransferase 1 (HPRT1) is a common housekeeping gene for sample normalization in the quantitative reverse transcriptase polymerase chain (qRT-PCR). However, co-amplification of HPRT1 pseudogenes may affect accurate results obtained in qRT-PCR. We designed a primer pair (HPSF) for pseudogene-free amplification of HPRT1 in qRT-PCR [1]. We showed specific amplification of HPRT1 mRNA in some common laboratory cell lines, including HeLa, NIH/3T3, CHO, BHK, COS-7 and VERO. This article provides data supporting the presence and location of HPRT1 pseudogenes within human and mouse genome, and the strategies used for designing primers that avoid the co-amplification of contaminating pseudogenes in qRT-PCR. In silico analysis of human genome showed three homologous sequences for HPRT1 on chromosomes 4, 5 and 11. The mRNA sequence of HPRT1 was aligned with the pseudogenes, and the primers were designed toward 5′ end of HPRT1 mRNA that was only specific to HPRT1 mRNA not to the pseudogenes. The standard curve plot generated by HPSF primers showed the correlation coefficient of 0.999 and the reaction efficiency of 99.5%. Our findings suggest that HPSF primers can be recommended as a candidate primer pair for accurate and reproducible qRT-PCR assays.

Lesch-Nyhan Syndrome in an Indian Child.

Hypoxanthine guanine phosphoribosyl transferase-1 (HGPRT-1) leading to Lesch-Nyhan syndrome (LNS) is one of the important causes of self-mutilation. Hereby, we report a case of LNS in a three and half-year-old male child, who presented with characteristic self-mutilating behavior. He had history of developmental delay, difficulty in social interaction, attention deficit and features of autism. His serum blood biochemistry was normal except for low hemoglobin levels and raised serum uric acid levels. With a diagnosis of LNS, the child was treated with allopurinol. With various modalities of physical restraint, his self-mutilating behavior came under control and currently the patient is being followed up.

Prenatal diagnosis based on HPRT1 gene mutation in a Lesch–Nyhan family

We explored the feasibility of applying gene diagnosis in prenatal diagnosis by analysis of hypoxanthine–guanine phosphoribosyltransferase-1 (HPRT1) gene mutation in a Chinese Lesch–Nyhan family. A homozygous mutation of p.R170X (c.508C＞T) in HPRT1 gene was detected in the proband, and a heterozygous mutation of p.R170X was detected in his mother. This mutation failed to be found in the 50 unrelated healthy individuals. Prenatal diagnosis indicated that the foetus was male and also carried p.R170X (c.508C＞T) mutation, same as the proband. Parents of the foetus decided termination of pregnancy, and the result of gene analysis for the aborted tissue was consistent with that of prenatal diagnosis. We can see that Lesch–Nyhan syndrome (LNS) is caused by non-sense mutation p.R170X（c.508C＞T）in HPRT1 gene in this family. Prenatal gene diagnosis is a valid strategy to prevent LNS because it can avoid the birth of LNS foetuses.

A Global Increase in 5‐Hydroxymethylcytosine Levels Marks Osteoarthritic Chondrocytes

To investigate the role of the newly discovered epigenetic mark 5-hydroxymethylcytosine (5hmC) and its regulators in altered gene expression in osteoarthritis (OA). Cartilage was obtained from OA patients undergoing total knee arthroplasty and from control patients undergoing anterior cruciate ligament reconstruction. Global levels of 5hmC and 5-methylcytosine (5mC) were investigated using immunoblotting, enzyme-linked immunosorbent assays, and cellular staining. Gene expression changes were monitored by quantitative polymerase chain reaction (PCR) analysis. Levels of locus-specific 5hmC and 5mC at CpG sites in the matrix metalloproteinase 1 (MMP-1), MMP-3, ADAMTS-5, and hypoxanthine guanine phosphoribosyltransferase 1 (HPRT-1) promoters were quantified using a glucosylation and enzyme digestion-based method followed by quantitative PCR analysis. Global and locus-specific 5hmC levels and gene expression changes were monitored in normal chondrocytes stimulated with inflammatory cytokines to identify the effect of joint inflammation. A global 5-6-fold increase in 5hmC concomitant with a loss of TET1 was observed in human OA chondrocytes compared to normal chondrocytes. Enrichment of 5hmC was observed in promoters of enzymes critical to OA pathology, MMP-1 and MMP-3. Short-term treatment of normal chondrocytes with inflammatory cytokines induced a rapid decrease in TET1 expression but no global or locus-specific 5hmC enrichment. This study provides the first evidence of an epigenetic imbalance of the 5hmC homeostasis in OA leading to TET1 down-regulation and 5hmC accumulation. Our experiments identify 5hmC and its regulators as potential diagnostic and therapeutic targets in OA.

Selection of Reliable Reference Genes for Real-time qRT-PCR Analysis of Zi Geese (<italic>Anser anser domestica</italic>) Gene Expression

Zi geese (Anser anser domestica) belong to the white geese and are excellent layers with a superior feed-to-egg conversion ratio. Quantitative gene expression analysis, such as Real-time qRT-PCR, will provide a good understanding of ovarian function during egg-laying and consequently improve egg production. However, we still don’t know what reference genes in geese, which show stable expression, should be used for such quantitative analysis. In order to reveal such reference genes, the stability of seven genes were tested in five tissues of Zi geese. Methodology/Principal Findings: The relative transcription levels of genes encoding hypoxanthine guanine phosphoribosyl transferase 1 (HPRT1), β-actin (ACTB), β-tubulin (TUB), glyceraldehyde-3-phosphate-dehydrogenase (GADPH), succinate dehydrogenase flavoprotein (SDH), 28S rRNA (28S) and 18S rRNA (18S) have been quantified in heart, liver, kidney, muscle and ovary in Zi geese respectively at different developmental stages (1 d, 2, 4, 6 and 8 months). The expression stability of these genes was analyzed using geNorm, NormFinder and BestKeeper software. Conclusions: The expression of 28S in heart, GAPDH in liver and ovary, ACTB in kidney and HPRT1 in muscle are the most stable genes as identified by the three different analysis methods. Thus, these genes are recommended for use as candidate reference genes to compare mRNA transcription in various developmental stages of geese.

Evaluation of potential reference genes for real time RT-PCR studies in Atlantic halibut (Hippoglossus Hippoglossus L.); during development, in tissues of healthy and NNV-injected fish, and in anterior kidney leucocytes

BackgroundReal time RT-PCR has become an important tool for analyzing gene expression in fish. Although several housekeeping genes have been evaluated in Atlantic halibut (Hippoglossus Hippoglossus L.), appropriate reference genes for low copy mRNA transcripts at the earliest developmental stages have not been identified. No attempts have been reported to identify suitable reference genes in halibut infected with NNV or in stimulated halibut leucocytes. In this study, β-actin1 (ACTB1), elongation factor 1 alpha (EF1A1), hypoxanthine-guanine phosphoribosyltransferase 1 (HPRT1), ribosomal protein L7 (RPL7), tubulin beta 2C (Tubb2C), and ubiquitin-conjugating enzyme (UbcE) were evaluated as reference genes for normalization of real time RT-PCR data during Atlantic halibut development, in tissue of healthy and NNV-infected fish, and in in vivo and in vitro stimulated anterior kidney leucocytes.ResultsThe expression of all six genes was relatively stable from the unfertilized egg until 12 day degrees post fertilization (ddpf). However, none of the selected genes were found to be stably expressed throughout halibut development. The mRNA levels of the six genes increased from 18 ddpf, when zygotic transcription is likely to be activated, and stabilized at different time points. The Excel-based software programs BestKeeper, geNorm, and NormFinder ranked EF1A1 and UbcE as the best candidate reference genes before activation of zygotic transcription, and RPL7 and EF1A1 as the best candidates after hatching. EF1A1 and RPL7 were also listed as the best reference genes when exploring the expression levels of the six genes in various halibut organs, both in non-injected fish and in mock- and NNV-injected fish. None of the reference genes were found optimal for normalization of real time RT-PCR data from in vitro stimulated anterior kidney leucocytes.ConclusionGenerally, it was found that EF1A1 and RPL7 were the genes that showed least variation, with HPRT1 and UbcE as intermediate genes, and ACTB1 and Tubb2C as the least stable ones. None of the six reference genes can be recommended as reference gene candidates in ConA-PMA stimulated leucocytes. However, UbcE can be a good candidate in other experimental setups. This study emphasizes the need for reference gene evaluation, as universal reference genes have not been identified.

Expression of X-linked genes in deceased neonates and surviving cloned female piglets.

Animal cloning through somatic cell nuclear transfer (NT) is very inefficient, probably due to insufficient reprogramming of the donor nuclei, which in turn would cause the dysregulation of gene expression. X-Chromosome inactivation (XCI) is a multi-step epigenetic process utilized by mammals to achieve dosage compensation in females. Our aim was to determine if any dysregulation of X-linked genes, which would be indicative of unfaithful reprogramming of donor nuclei, was present in cloned pigs. Real time reverse transcription polymerase chain reaction (RT-PCR) was performed to quantify the transcript levels of five X-linked genes, X inactivation-specific transcript (XIST), TSIX (the reverse spelling of XIST), hypoxanthine guanine phosphoribosyltransferase 1 (HPRT1), glucose-6-phosphate dehydrogenase (G6PD), V-raf murine sarcoma 3,611 viral oncogene homolog 1 (ARAF1), and one autosomal gene, alpha-1 type IV collagen (COL4A1) in major organs of neonatal deceased and surviving female cloned pigs as well as their age-matched control pigs from conventional breeding. Aberrant expression level of these genes was prevalent in the neonatal deceased clones, while it was only moderate in cloned pigs that survived after birth. These results suggest a correlation between the viability of the clones and the normality of their gene expression and provide a possible explanation for the death of a large portion of cloned animals around birth.

Validation of two reference genes for mRNA level studies of murine disease models in neurobiology

Reverse transcription of extracted cellular RNA combined with real-time PCR is now an established method for sensitive detection and quantification of specific mRNA level changes in experimental models of neurological diseases. To neutralize the impact of experimental error and make quantification more precise, normalization of test gene data using data from a constantly expressed gene, a reference gene that is tested along with the test gene, is required. There is no single gene constantly expressed under all experimental conditions. For a given set of conditions or a given disease model, identification of an unaffected reference gene is necessary. In this report, we present our findings from evaluation and validation of the genes encoding hypoxanthine guanine phosphoribosyl transferase 1 (HPRT1) and glyceraldehyde phosphate dehydrogenase (GAPDH) as individual reference genes in mRNA level studies involving four murine neurological disease models. We find both genes are suitable as a reference gene with these four models, provided quantification of subtle changes are avoided. We furthermore demonstrate that above a certain threshold of test mRNA level changes and given high quality RNA processing, normalization to total RNA alone provides for equally reliable quantitative mRNA level results.