OBJECTIVETo perform the genetic identification of cloche(172) mutant zebrafish.

METHODSThe chemical mutagen N-ethyl-N-nitrosourea (ENU) was used to treat the AB stain male fish. Large-scale forward genetic screening was carried out to search for lyC-deficient zebrafish mutant by WISH. The morphology changes of the embryos at 3 days postfertilization (3dpf) stage were observed and the cloche(172) gene was identified by mapping and complementation test.

RESULTSWe selected 4 lyC-deficient zebrafish by WISH. cloche(172) mutant showed morphological changes similar to cloche mutant in 3dpf stage. One fourth of the embryos showed cloche phenotype as found in complementation test, and the cloche(172) gene was mapped on the telomere of zebrafish 13 chromosome where cloche gene was located. Numerous red blood cells were observed in the cloche(172) mutant, while only a few cells were found in the cloche mutant in the tail region by o-dianisdine staining.

CONCLUSIONcloche(172) gene which is responsible for the phenotype of cloche mutant may be a novel point mutation allele of the cloche mutant.

Alleles ; Animals ; Chromosome Mapping ; Cloning, Molecular ; Embryo, Nonmammalian ; embryology ; metabolism ; Ethylnitrosourea ; toxicity ; Genetic Complementation Test ; Male ; Muramidase ; genetics ; Mutation ; Zebrafish ; embryology ; genetics ; Zebrafish Proteins ; genetics

The genome instability and tumorigenicity of induced pluripotent stem cells (iPSC) hinder their great potentials for clinical application. Using episomal vectors to generate iPSC is the best way to solve safety issues at present. This method is simple and the exogenous gene was not integrated into the host genome. However, the reprogramming efficiency for this method is very low and thus limits its usage. This study was purposed to improve episomal method for generating induced pluripotent stem cells from cord blood mononuclear cells (CB MNC), to establish integration-free iPSC technology system, and to lay the foundation for individualized iPSC for future clinical uses. To improve the reprogramming efficiency for iPSC, episomal method was used at various combinations of episomal vectors, pre-stimulating culture mediums and oxygen condition were tested to optimize the method. The results showed that using erythroid culture medium for culturing 8 days, transfecting with episomal vectors with SFFV (spleen focus forming virus) promoter under the hypoxic condition (3%), CB MNC could be mostly efficiently reprogrammed with the efficiency 0.12%. Furthermore, the results showed that erythroblasts (CD36(+)CD71(+)CD235a(low)) were the cells that are reprogrammed with high efficiency after culture for 8 days. It is concluded that a highly efficient and safe method for generation of integration-free iPSC is successfully established, which is useable in clinical study.
Cell Culture Techniques ; methods ; Cellular Reprogramming ; Genetic Vectors ; Humans ; Induced Pluripotent Stem Cells ; cytology ; Plasmids ; Transfection

Nowadays genetic tests are available in a growing number of countries, for an expanding set of conditions. Nonetheless, many Chinese people are still not familiar with the principle, testing types, technologies used in this process, application and benefits to society, and national or international administration of genetic testing. It is essential that this increased use of genetic testing should be accompanied by appropriate oversight. This article has roughly reviewed the proceeding of genetic testing these years, which will help us learn more about the new coming era of human genetics and molecular medical revolutions.
Genetic Predisposition to Disease ; prevention & control ; Genetic Techniques ; trends ; Genetic Testing ; trends ; Humans

Since Yamanaka successfully reprogrammed murine fibroblasts into iPSCs in 2006, iPSCs technology has drawn much attention worldwide. Although iPSCs provides tremendous possibilities for both basic research and regenerative medicine, it has meanwhile potential risks, e.g. tumorigenicity. Scientists, therefore, have made efforts in clarifying the mechanism of the cause for iPSCs tumorigenicity and the way how to reduce the risk. The results of some researches reveal some of tumorigenic factors, e.g. the partial similarity of gene expression profiles between cancer cells and iPSCs, the accumulation of the genetic damages in the course of reprogramming process, and mutation in the cellular culture. As a consequence, numerous methods for reducing iPSCs tumorigenicity have been explored, such as minimized use of the reprogramming factors at the controlled manner, and the selection of the expression vector or parental cells. In this paper, the cause of iPSCs tumorigenicity and the current achievements on preventing iPSCs tumorigenesis are reviewed.
Animals ; Carcinogenesis ; Cell Culture Techniques ; Cellular Reprogramming ; Fibroblasts ; Genetic Vectors ; Humans ; Induced Pluripotent Stem Cells ; cytology ; Mice ; Mutation ; Transcriptome

Humans ; Child ; Communicable Diseases/therapy* ; Molecular Diagnostic Techniques ; Genetic Testing

Gene Transfer Techniques ; Genetic Engineering ; Genetic Vectors ; Humans ; Serum Albumin ; chemistry ; genetics

Clostridium acetobutylicum, a biofuel-butanol producer, has attracted worldwide interests. Strain improvement is important for the process of biobutanol industrialization where efficient genetic modification systems are essential. In this review, the history of genetic modification systems of C. acetobutylicum was introduced, and the types and principles of these systems and their disadvantages are summarized and analysed. The development of updated genetic modification systems for C. acetobutylicum is also proposed.
Biofuels ; Butanols ; analysis ; metabolism ; Clostridium acetobutylicum ; genetics ; Gene Expression Regulation, Bacterial ; Genetic Engineering ; Genetic Techniques

Knockout is an important method for gene function study, while vector is the core of gene knockout. In order to obtain an effective vector for rapid construction of mutant and essentiality identification of the corresponding gene, we constructed a recombinant plasmid named plDM-T based on the temperature-sensitive and replication- defective plasmid plDM1 by inserting an Xcm I adapter into the EcoR I and Pst I sites of pIDM1. Digesting with Xcm I, pIDM-T can be prepared as a linear T-vector for PCR products cloning and be used to knockout the corresponding gene in the genome with insertion duplication mutagenesis. After the verification of temperature sensitivity of the replication of the plasmid, we cloned two Salmonella pullorum genes eno and ybdr into the constructed pIDM-T, and two recombinant plasmids pIDM-T_eno and pIDM-T_ybdr were identified. The recombinant plasmids were then transformed into S. pullorum strain CVCC527 and the IPC (Integration rate per cell) values were calculated. As a result, we identified the eno gene as an essential gene and the ybdr as a non-essential gene in CVCC527. We verified the correctness of recombination site in ybdr recombinant 527 clones (Sal delta ybdr) by PCR and sequencing. The pIDM-T vector can be used for gene knockout in S. pullorum, as well as the identification of essentiality of the corresponding genes, which offers an effective and rapid tool for gene function study in Salmonella.
Cloning, Molecular ; Gene Knockout Techniques ; Genetic Vectors ; genetics ; metabolism ; Plasmids ; genetics ; Recombinant Proteins ; genetics ; metabolism ; Recombination, Genetic ; Salmonella ; classification ; genetics

Communicable Diseases ; genetics ; Genetic Diseases, Inborn ; genetics ; Genetic Testing ; Genotype ; Genotyping Techniques ; Humans ; Molecular Diagnostic Techniques ; Precision Medicine ; methods ; trends

OBJECTIVE: To explore the methods to detect and intervene children's late-onset hearing loss early which are suitable for basic-level hospitals. METHOD: Udiology and imaging diagnosis had been given to the children who passed the newborn hearing screening but showed auditory behavior disorders in the growth process, and individualized interventions were given according to the results of diagnosis. Seven children with high risk for hereditary deafness were sent to superior hospital and had molecular screening of common mutations of inherited deafness carried out, then corresponding prevention guidance and intervention were given to them. RESULT: Fifty-two cases with late-onset hearing loss or verbal disorders were detected by auditory behavior observations,including 4 cases of auditory neuropathy, 4 cases of unilateral sensorineural deafness, 27 cases of secretory otitis media. 13 cases of bilateral sensorineural deafness and 4 cases of autism. Seven newborns with high risk of hereditary deafness were sent to the Third Affiliated Hospital of Sun Yat-Sen University and received molecular screening of common mutations of inherited deafness. One case with GJB2 compound heterozygous mutations was detected and followed up to 4 years old, he was found bilateral moderate hearing loss and accepted the hearing aids at 2 years old. Mitochondrial DNA 1555 a > G heterogeneity mutation in 2 cases and GJB2 235 delC single heterozygous mutations in 3 cases, no mutation in 1 case, all these 6 cases have been followed-up until now, their hearing are normal. CONCLUSION Children's auditory behavior observations and the superior hospitals referral performing high risk individual screening for newborns with high risk for hereditary deafness can detect children's late-onset hearing loss in time, this model is suitable for basic-level hospitals.
Behavior Observation Techniques ; Child ; Connexin 26 ; Connexins ; genetics ; DNA Mutational Analysis ; DNA, Mitochondrial ; genetics ; Deafness ; diagnosis ; genetics ; Early Diagnosis ; Genetic Testing ; Hearing Tests ; Heterozygote ; Humans ; Infant, Newborn ; Male ; Mutation