Subtractive phage display technology identifies zebrafish marcksb that is required for gastrulation.

In the present study, we used a phage display technique to screen differentially expressed proteins from zebrafish post-gastrula embryos. With a subtractive screening approach, 6 types of single-chain Fv fragments (scFvs) were screened out from an scFv antibody phage display library by biopanning against zebrafish embryonic homogenate. Four scFv fragments (scFv1, scFv3, scFv4 and scFv6) showed significantly stronger binding to the tailbud embryos than to the 30%-epiboly embryos.

Antigenic analysis of grass carp reovirus using single-chain variable fragment antibody against IgM from Ctenopharyngodon idella.

Grass carp (Ctenopharyngodon idella) is an important species of freshwater aquaculture fish in China. However, grass carp reovirus (GCRV) can cause fatal hemorrhagic disease in yearling populations. Until now, a strategy to define the antigenic capacity of the virus's structural proteins for preparing an effective vaccine has not been available.

[Linkage analysis of susceptibility loci in 2 target chromosomes in pedigrees with paranoid schizophrenia and undifferentiated schizophrenia].

Objective: To investigate the relationship of susceptibility loci in chromosomes 1q21-25 and 6p21-25 and schizophrenia subtypes in Chinese population.

Methods: A genomic scan and parametric and non-parametric analyses were performed on 242 individuals from 36 schizophrenia pedigrees, including 19 paranoid schizophrenia and 17 undifferentiated schizophrenia pedigrees, from Henan province of China using 5 microsatellite markers in the chromosome region 1q21-25 and 8 microsatellite markers in the chromosome region 6p21-25, which were the candidates of previous studies. All affected subjects were diagnosed and typed according to the criteria of the Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition, Text Revised (DSM-IV-TR; American Psychiatric Association, 2000).

Screening of LRRK2 interactants by yeast 2-hybrid analysis.

Objective: To isolate and identify the potential binding partners of LRRK2, a gene linked to both dominant familial form and sporadic form of Parkinson's disease, thus to further our knowledge of its function.

Methods: We used a sequence containing full-length of COR domain and part of ROC and MAPKKK domain as bait. The bait amplified by polymerase chain reaction (PCR) was then cloned into a yeast expression plasmid pGBKT7.

Application of methyl parathion hydrolase (MPH) as a labeling enzyme.

Methyl parathion hydrolase (MPH) is an enzyme that catalyzes the degradation of methyl parathion, generating a yellow product with specific absorption at 405 nm. The application of MPH as a new labeling enzyme was illustrated in this study. The key advantages of using MPH as a labeling enzyme are as follows: (1) unlike alkaline phosphatase (AP), horseradish peroxidase (HRP), and glucose oxidase (GOD), MPH is rarely found in animal cells, and it therefore produces less background noise; (2) its active form in solution is the monomer, with a molecular weight of 37 kDa; (3) its turnover number is 114.

Efficient protein expression from the endogenous RNA polymerase I promoter using a human ribosomal DNA targeting vector.

Vector systems to deliver, integrate and express therapeutic genes in host cells are essential for gene therapy. In the present study, we investigated a novel vector system for integration and expression of a transgene. In this system, the transgene expression was driven by an endogenous RNA polymerase I (Pol I) promoter after being integrated into the ribosomal DNA (rDNA) locus.

[Mapping of pathogenic genes in a pedigree with autosomal dominant ichthyosis vulgaris].

Objective: To elucidate the pathogenic genes in a pedigree with autosomal dominant ichthyosis vulgaris (IV).

Methods: Linkage analysis was performed by using STR markers in chromosome 1, and mutation detection was used to screen for FLG gene mutation.

Results: A maximum two-point Lod score of 3.

[Biological characteristics and safety evaluation of endothelial progenitor cells from the umbilical cord blood].

Objective: To investigate the biological characteristics of endothelial progenitor cells (EPCs) from the umbilical cord blood (UCB), and to evaluate their oncogenicity after long-term culture in vitro.

Methods: The mononuclear cells (MNCs) were isolated from the UCB and cultured in MCDB131 medium supplemented with 20% FBS, VEGF and other growth factors. Morphology of the EPCs was observed, and the growth curve of the EPCs was investigated.

[Identification of the origin of marker chromosome by comparative genomic hybridization].

Objective: To identify the origin of the marker chromosome in a patient with chromosome aberration, and to provide the precise genetic diagnosis.

Methods: Comparative genomic hybridization (CGH) and fluorescence in situ hybridization (FISH) were performed to detect the known small marker chromosome in this patient.

Results: The small marker chromosome originated from chromosome 13 pter->q12.

[Preimplantation genetic diagnosis of Duchenne muscular dystrophy by single cell triplex PCR].

Objective: To detect two exons of Duchenne muscular dystrophy (DMD) gene and a gender discrimination locus amelogenin gene by single cell triplex PCR, and to evaluate the possibility of this technique for preimplantation genetic diagnosis (PGD) in DMD family with DMD deletion mutation.

Methods: Single lymphocytes from a normal male, a normal female, two DMD patients (exon 8 and 47 deleted, respectively) and single blastomeres from the couples treated by the in vitro fertilization pre-embryo transfer (IVF-ET) and without family history of DMD were obtained. Exons 8 and 47 of DMD gene were amplified by a triplex PCR assay, the amelogenin gene on X and Y chromosomes were co-amplified to analyze the correlation between embryo gender and deletion status.

[Azoospermia factor microdeletion on Y chromosome in patients with idiopathic azoospermia or severe oligozoospermia].

Objective: To investigate the correlation between male infertility and Y chromosome microdeletions of azoospermia factor (AZF) regions, and to establish a reliable genetic diagnosis in idiopathic infertile male patients with azoospermia or severe oligozoospermia.

Methods: Multiplex PCR amplification of 6 sequence-tagged sites in AZF regions of the Y chromosome was examined among 100 normal karyotype male patients with azoospermia or oligozoospermia.

Results: Four patients (4%) had Y chromosome microdeletions, the microdeletions of 3 patients were idiopathic azoospermic and those of the other 1 patient were secretory azoospermia.

[Expression of single chain fragment variable P1D3 antibody against shrimp white spot syndrome virus in Pichia pastoris].

White spot syndrome virus (WSSV) is a major pathogen in aquaculture penaeid shrimp, which caused catastrophic economic losses in the worldwide. No adequate treatments against WSSV are available. In order to study infection mechanism of WSSV, a phage display scFv cDNA library against WSSV was constructed and a neutralizing antibody of scFv P1D3 was selected in our lab previously.

[Confirmation of the extra small chromosome in abnormality karyotype by PCR and FISH].

Objective: To investigate the source of the extra small chromosome in a patient with karyotype 45,X[115]/46,X + mar[45]/46,XY[29].

Methods: The SRYgene was detected by PCR, and the chromosome Y probe that labeled with biotin was detected by fluorescence in situ hybridization.

Results: SRY gene is detected positive and the mar chromosome showed positive signal with FISH in human chromosome Y probe pool.

[A mutation 1633-26(C-->A) in EXT1 gene causes multiple exostoses].

Objective: To study the gene mutation in a patient with multiple exostoses, identify the disease-causing gene mutation.

Methods: Polymerase chain reaction and DNA sequencing were used to screen the EXT1 or EXT2 gene mutation, while mismatch primer amplification and restriction endonuclease digestion were performed to confirm the mutation.

Results: By DNA sequencing, a mutation in the seventh intron was detected and located at 26 bp of 3' splice site upstream in EXT1 gene, which was unreported before.

[Hotspot of the mutations of keratin 9 gene in a diffuse palmoplantar keratoderma family].

Objective: To identify the gene causing diffuse palmoplantar keratoderma in a Chinese pedigree.

Methods: Four normal individuals and 3 patients in a diffuse palmoplantar keratoderma family and 10 unrelated control samples were recruited. The hotspot of the mutations of keratin 9 gene was analyzed by polymerase chain reaction and direct sequencing.

A minidystrophin-EGFP fusion gene expressed in Cos-7 cells mediated by human source vector.

Objective: To construct a human source vector containing minidystrophin-EGFP fusion gene and investigate its expression in Cos-7 cells.

Methods: The recombinant human source vector named pHrnDysG was constructed with PCR-clone methods. Three fragments of dystrophin gene were PCR amplified from normal human dystrophin gene cDNA (GenBank NM04006).

[Prenatal diagnosis of prelingual deafness by determination of SLC26A4 gene mutation].

Objective: To identify deafness related gene and provide its prenatal diagnosis to avoid deaf fetus delivery.

Methods: DNA was extracted from amniotic cells in a pregnant woman close to 21 weeks' gestation, as well as from peripheral blood cells of the pregnant woman, her husband and their two sons. Screening for GJB2 and SLC26A4 gene mutations was firstly performed in the deafness proband (the first son of the couple), and then it was carried out in the fetus and the rest family members.

[Infrequent X chromosome abnormality and X-linked syndromic deafness].

Objective: To make clear the relationship between the X chromosome abnormality and sydromic deafness through genetic analysis of a pedigree with X-linked syndromic deafness.

Methods: The chromosome number and structure of the family members were analyzed by the standard and high resolution banding with Giemsa, and fluorescent in situ hybridization. The allelic number of the DNA segment in X chromosome was studied with genetic markers.

Molecular analysis of SLC26A4 gene in a Chinese deafness family.

Objective: To identify the pathogenic gene for a non-syndromic hearing loss family.

Methods: Mutation analysis was carried out by polymerase chain reaction and direct sequencing of all exons of SLC26A4 (solute carrier family 26, member 4) gene.

Results: Compound heterozygous mutations N392Y and S448X were detected in the proband of the family, heterozygous mutation S448X was detected in the father, heterozygous mutation N392Y was detected in the mother.

Intracellular distribution, assembly and effect of disease-associated connexin 31 mutants in HeLa cells.

Mutations in connexin 31 (Cx31) are associated with erythrokeratodermia variabilis (EKV), hearing impairment and peripheral neuropathy; however, the pathological mechanism of Cx31 mutants remains unknown. This study analyzed 11 disease-associated Cx31 variants and one non-disease-associated Cx31 variant and compared their intracellular distribution and assembly in HeLa cells and their effect on these cells. The fluorescent localization assay showed no gap junction plaque formation in the cells expressing the recessive EKV-associated mutant (L34P) and four hearing impairment-associated mutants (66delD, 141delI, R180X and E183K), significantly reduced plaque formation in the cells with five EKV-associated dominant mutants (G12R, G12D, R42P, C86S and F137L) and no obvious change in the cells with two other mutants (I141V and 652del12).

Cx31 is assembled and trafficked to cell surface by ER-Golgi pathway and degraded by proteasomal or lysosomal pathways.

Gap junctions, consisting of connexins, allow the exchange of small molecules (less than 1 KD) between adjacent cells, thus providing a mechanism for synchronizing the responses of groups of cells to environmental stimuli. Connexin 31 is a member of the connexin family. Mutations on connexin 31 are associated with erythrokeratodermia variabilis, hearing impairment and peripheral neuropathy.

Identification of the alternative promoters of the KChIP4 subfamily.

The subfamily of voltage-dependent potassium (Kv) channel interacting protein 4 (KChIP4) is made up of the auxiliary interacting protein of voltage-dependent potassium channels. In this study, the structure of four splicing variants of the human KChIP4 gene was analyzed. Three of the four isoforms of the KChIP4 gene, KChIP4.