Biomedical subjects

Yu-zhi Zhang

Publications and source records attributed to Yu-zhi Zhang.

4 recordsLinked to original sources

PubMed2006

[Knocking down rat Mecp2 expression by RNAi].

OBJECTIVE: To find the valid siRNA (small interference RNA) sequence to knock down rat Mecp2 expression for the analysis of Mecp2 function by RNA interference (RNAi). METHODS: A plasmid (pMecp2-RFP) expressing rat Mecp2 and marker gene red fluorescent protein (RFP) as a fusion gene was constracted. We first selected a candidate valid sequence by cotransfecting the pMecp2-RFP with 4 candidate siRNAs targeting rat Mecp2 to HEK293T cells respectively, and then identified the RNAi efficiency of the candidate valid siRNA by detecting its effect on suppressing the expression of endogenous rat Mecp2 in PC12 cells. RESULTS: siRNA sequence (5'-GCUGUGAAGGAAUCUUCUA-3') targeting rat Mecp2 mRNA 918-936nt was the valid sequence to knock down rat Mecp2 expression by RNA interference. As the target sequence was located in exon 4 of rat Mecp2 mRNA, we assumed that it could suppress the expression of Mecp2alpha and Mecp2beta at the same time. And as the target sequence was located in the coding region of rat Mecp2, we assumed that it could suppress the expression of both 1.9 kb and 10 kb of rat Mecp2 transcript. CONCLUSION: This study has laid the foundation for constructing rat Mecp2 knocked-down neuronal cell model to study the gene function of Mecp2 in brain development.

Animals↗

PubMed2006

[MECP2 gene mutations in twenty-six cases with atypical Rett syndrome].

OBJECTIVE: Rett syndrome (RTT) is an X-linked progressive neurodeveopmental disorder that almost exclusively affects girls, and is one of the most common causes of mental retardation in females, with an estimated prevalence of approximately 1 in 10,000 - 15,000 female individuals. Mutations in X-linked methyl-CpG-binding protein 2 (MECP2) gene, located on chromosome Xq28, have been found to be a cause of RS. A lot of mutations have been reported to be related to RS recently. Mutations are found in 70% - 85% of patients with classical RTT and in less than 50% of patients with atypical RS. Up to now, RTT is diagnosed based on a consistent counseling for clinical features and the established diagnostic criteria. The present study aimed to investigate frequency and type of mutation of MECP2 gene and if hot spot of mutation exits in patients with atypical RTT and find out the relationship between genotype and phenotype. METHODS: A systematic analysis of the entire coding region of MECP2 in 26 unrelated patients with atypical RTT was performed by polymerase chain reaction (PCR) and direct sequencing. Genomic DNA was extracted using standard procedures from the peripheral blood leukocytes of each patient. PCR amplification products were checked by 2% agarose gel electrophoresis and were subsequently sequenced with ABI 3730 Automated DNA Sequencer with both the forward and reverse primers. Mutational analyses were performed using normal human genomic MECP2 sequence as a reference (GenBank accession NO.AF030876). RESULTS: Seven mutations were identified in 12 of 26 patients. Most of the mutations were missense mutation; c.397C > T (R133C) was found in 3 of 26 patients; c.473C > T (T158M) and c.916C > T (R306C) were found in 2 of 26 patients, respectively; c.397A > G (R133H) and c.1005G > A (R335C) were found in 1 of 26 patients, respectively. One base pair deletion mutation (806delG) resulting in frameshift was found in 2 of 26 patients, and 1 base pair transversion at splice accept-site (IVS3-2A > T). CONCLUSION: The results of this study indicated that c.397C > T (R133C), c.473C > T (T158M) and c.916C > T (R306C) were hot spot mutations in MECP2 gene of patients with atypical RTT. There was some relationship between genotype and phenotype.

Base Sequence↗

PubMed2005

[Helper-virus-free herpes simplex virus-1 vector mediated LacZ gene expression in cultured cortical neurons].

OBJECTIVE: To obtain knowledge about the helper-virus-free packaging system of HSV-1 vector and its use in cultured neurons by investigating helper-virus-free packaging of HSV-1 vector and LacZ gene expression mediated by the vector in rat cultured cortical neurons. METHODS: The cosmids with inserts of HSV-1 genome were digested by Pac I, purified and cotransfected with HSV plasmid vector DNA (with LacZ gene) into 2-2 cells. After 3 hour incubation at 37 degrees C, the media were changed into DMEM with 6% CCS. And the 2-2 cells were incubated for 72 hours at 34 degrees C. Then the virus particles were harvested. The medium with the virus particles was added to BHK cells. After 24 hour further culture, X-gal staining was performed. The virus particles were added to 3 day cultured cortical neurons. After 1, 7 and 14 day further cultures, neurons were stained by X-gal and observed under microscope. RESULTS: The BHK cells (stained blue) expressing LacZ gene could be seen after 24 hour further culture. After 1, 7 and 14 day further cultures, neurons stained blue by X-gal staining could be seen all the time in different groups. CONCLUSION: Helper-virus-free package system can produce effective virus particles from HSV-1 plasmid vector with A tyrosine TH-NF chimeric promoter and mediate stable exogenous gene expression in cultured neurons, thus providing a useful tool for gene transfer and study of gene function in the nervous system.

Animals↗

PubMed2004

[Expression of mythl-CpG-binding protein-2 gene in developing rat brain].

OBJECTIVE: To analyze the expression of mecp2 gene at mRNA and protein;Cerebral cortex level in the cerebral cortex of the normal Wistar rat throughout development. METHODS: We chose the 15th day (E15), 17th day (E17), 19th day (E19) of embryo period, the day of birth (P0), the 7th day (P7), the 14th day (P14), the 28th day (P28) of postnatal period, and adulthood as analyzing time points. The expression of mecp2 gene at mRNA level was analyzed by real-time PCR and Northern blot. The expression of MeCP2 protein was analyzed by Western blot. RESULTS: There was one type of mecp2 mRNA transcript (approximately 10 kb) expressed in the cerebral cortex of the normal Wistar rat. The expression level of mecp2 mRNA varied subtly during the development. There was one type of MeCP2 protein (75 000) expressed in the cerebral cortex of the normal Wistar rat. The expression level of MeCP2 protein remained the lowest on E15, from E19 to adulthood the expression levels of MeCP2 protein increased dramatically compared with that on E15. From P7 to adulthood, the differences of expression between two time points were subtle. CONCLUSION: The expression level of MeCP2 protein increases as the neurons in the cerebral cortex of normal Wistar rat grow mature. This indicates that MeCP2 protein is very important to neuron's maturation, and probably has relationship with maintaining maturation state of neurons.

Animals↗