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1.
Protein & Cell ; (12): 39-56, 2021.
Article in English | WPRIM | ID: wpr-880896

ABSTRACT

Gene expression labeling and conditional manipulation of gene function are important for elaborate dissection of gene function. However, contemporary generation of pairwise dual-function knockin alleles to achieve both conditional and geno-tagging effects with a single donor has not been reported. Here we first developed a strategy based on a flipping donor named FoRe to generate conditional knockout alleles coupled with fluorescent allele-labeling through NHEJ-mediated unidirectional targeted insertion in zebrafish facilitated by the CRISPR/Cas system. We demonstrated the feasibility of this strategy at sox10 and isl1 loci, and successfully achieved Cre-induced conditional knockout of target gene function and simultaneous switch of the fluorescent reporter, allowing generation of genetic mosaics for lineage tracing. We then improved the donor design enabling efficient one-step bidirectional knockin to generate paired positive and negative conditional alleles, both tagged with two different fluorescent reporters. By introducing Cre recombinase, these alleles could be used to achieve both conditional knockout and conditional gene restoration in parallel; furthermore, differential fluorescent labeling of the positive and negative alleles enables simple, early and efficient real-time discrimination of individual live embryos bearing different genotypes prior to the emergence of morphologically visible phenotypes. We named our improved donor as Bi-FoRe and demonstrated its feasibility at the sox10 locus. Furthermore, we eliminated the undesirable bacterial backbone in the donor using minicircle DNA technology. Our system could easily be expanded for other applications or to other organisms, and coupling fluorescent labeling of gene expression and conditional manipulation of gene function will provide unique opportunities to fully reveal the power of emerging single-cell sequencing technologies.


Subject(s)
Alleles , Animals , CRISPR-Cas Systems , DNA End-Joining Repair , DNA, Circular/metabolism , Embryo, Nonmammalian , Gene Editing/methods , Gene Knock-In Techniques , Gene Knockout Techniques , Genes, Reporter , Genetic Loci , Genotyping Techniques , Green Fluorescent Proteins/metabolism , Integrases/metabolism , Luminescent Proteins/metabolism , Mutagenesis, Insertional , Single-Cell Analysis , Zebrafish/metabolism
2.
Gac. méd. Méx ; 155(5): 463-470, Sep.-Oct. 2019. tab, graf
Article in English | LILACS | ID: biblio-1286544

ABSTRACT

The first draft of the human genome sequencing published in 2001 reported a large number of single nucleotide polymorphisms (SNPs). Given that these polymorphisms could practically represent all the variability involved in the susceptibility, protection, severity, among other aspects, of various common diseases, as well as in their response to medications, it was thought that they might be “the biomarkers of choice” in personalized genomic medicine. With the new information obtained from the sequencing of a larger number of genomes, we have understood that SNPs are only an important part of the genetic markers involved in these traits. In addition to SNPs, other variants have been identified, such as insertions/deletions (INDELs) and copy number variants (CNVs), which – in addition to classic variable number tandem repeats (VNTRs) and short tandem repeats (STRs) – originate or contribute to the development of diseases. The use of these markers has served to identify regions of the genome involved in Mendelian diseases (one gene-one disease) or genes directly associated with multifactorial diseases. This review has the purpose to describe the role of STRs, VNTRs, SNPs, CNVs and INDELs in linkage and association studies and their role in Mendelian and multifactorial diseases.


Subject(s)
Humans , Genetic Variation/physiology , Disease/genetics , Polymorphism, Single Nucleotide , Genetic Markers , Genome, Human , Mutagenesis, Insertional , Gene Deletion , Tandem Repeat Sequences , Lod Score , Mutation
3.
Rio de Janeiro; s.n; 2019. xiv, 152 p. ilus.
Thesis in Portuguese | LILACS | ID: biblio-1049943

ABSTRACT

Celulases fúngicas têm sido usadas para degradar a biomassa lignocelulósica para a produção de bioetanol. Celulases industriais como Cel7A de Trichoderma reesei (TrCel7A) são críticas neste processo. A compreensão da estrutura e dinâmica é crucial para a reengenharia da atividade celulolítica. Esta enzima é formada por dois domínios ligados por um linker flexível e altamente glicosilado. No entanto, a flexibilidade do linker tem dificultado a determinação da estrutura completa da Cel7A. Assim, na ausência de dados experimentais de alta resolução, aplicamos a modelagem integrativa para construir um modelo da enzima completa. Em seguida, estudamos os efeitos da glicosilação na estrutura e dinâmica da apo TrCel7A por meio de simulações. A análise da dinâmica essencial mostrou que a O-glicosilação no linker levou à estabilização da dinâmica global da proteína. Os glicanos O-ligados parecem restringir a distribuição dos ângulos diedros desta região, selecionando conformações mais alongadas. Além da flexibilidade reduzida, os movimentos interdomínios funcionais foram preservados no sistema glicosilado. Em contraste, observamos grande plasticidade conformacional na ausência de glicosilação, mas os domínios funcionais frequentemente colapsaram. Nós relatamos aqui evidências de que a flexibilidade dirigida no linker de Cel7A por mutações pontuais, incluindo modificações de sítios de glicosilação, poderia ser uma estratégia promissora para melhorar a atividade da celulase. (AU)


Subject(s)
Humans , Trichoderma , Glycosylation , Mutagenesis, Insertional , Cellulases
4.
Article in Chinese | WPRIM | ID: wpr-771515

ABSTRACT

Ralstonia solanacearum strain PRS-84 used in this study was isolated from diseased Pogostemon cablin plants in our previous study.The competent cells of R.solanacearum strain PRS-84 were transformed by electroporation with Tn5 transposon and then were plated on TTC agar plates containing kanamycin to select for kanamycin-resistant colonies.The detection of kanamycin-resistant gene in kanamycin-resistant colonies was performed by PCR.Further,the flanking fragments of Tn5 transposon insertion site in the mutants were amplified by inverse PCR,and the flanking fragments were sequenced and analyzed.The results indicated that the kanamycin-resistant colonies were obtained in the transformation experiment of R.solanacearum strain PRS-84 by electroporation with Tn5 transposon.A specific band of approximately 700 bp was amplified by PCR from kanamycin-resistant colonies.The flanking sequences of Tn5 transposon insertion site in the transformants were obtained by inverse PCR.After sequencing and sequence analysis of Tn5 transposon insertion site in mutants,we preliminarily speculated that the Tn5 transposon inserted in the typ A gene,rec O gene and gid A gene in three mutants,respectively.A random mutagenesis system of R.solanacearum strain PRS-84 by electroporation with Tn5 transposon has been established,and the Tn5 insertion mutants have been obtained.This study might facilitate the creation of mutant library and the discovery of the virulence gene of R.solanacearum isolated from P.cablin.


Subject(s)
DNA Transposable Elements , Electroporation , Genes, Bacterial , Mutagenesis, Insertional , Pogostemon , Microbiology , Ralstonia solanacearum , Genetics , Virulence
5.
Article in English | WPRIM | ID: wpr-763132

ABSTRACT

PURPOSE: Epidermal growth factor receptor (EGFR) exon 20 insertion mutations account for approximately 4% of all EGFR mutations. Given the rarity of this mutation, its clinical outcomes are not fully established. MATERIALS AND METHODS: Between 2009 and 2017, non-small cell lung cancer (NSCLC) patients who showed an exon 20 insertion were retrospectively reviewed for clinical characteristics and outcomes, including responses to chemotherapy (CTx) or targeted therapy. RESULTS: Of 3,539 NSCLC patients who harbored an activating EGFR mutation, 56 (1.6%) had an exon 20 insertion. Of the advanced NSCLC patients, 27 of 1,479 (1.8%) had an exon 20 insertion. The median overall survival was 29.4 months (95% confidence interval 9.3 to 49.6) for 27 advancedNSCLC patients. The 22 patientswho received systemic CTx achieved a 50.0% response rate and a 77.2% disease control rate, with 4.2 months of progression-free survival. Six patients received EGFR tyrosine kinase inhibitors (TKIs). Three of the four patients that had only an exon 20 insertion showed progressive disease, while one showed stable disease. The othertwo patients had an exon 20 insertion and another EGFR mutation and achieved a partial response. CONCLUSION: The incidence of an exon 20 insertion mutation is rare in Korea and occasionally accompanied by other common EGFR mutations. Although the response to systemic CTx. in these patients is comparable to that of patients with other mutations, the response rate to first- or second-generation EGFR TKIs is quite low. Therefore, the development of a more efficient agent is urgently needed.


Subject(s)
Carcinoma, Non-Small-Cell Lung , Disease-Free Survival , Drug Therapy , Exons , Humans , Incidence , Korea , Mutagenesis, Insertional , Protein-Tyrosine Kinases , ErbB Receptors , Retrospective Studies
6.
Arch. endocrinol. metab. (Online) ; 62(1): 21-26, Jan.-Feb. 2018. tab
Article in English | LILACS | ID: biblio-887636

ABSTRACT

ABSTRACT Objectives This study aimed to evaluate the frequencies of the angiotensin converting enzyme (ACE) gene insertion/deletion (I/D) and methylenetetrahydrofolate reductase (MTHFR) gene C677T polymorphisms in obese patients with and without type 2 diabetes mellitus (T2DM). Subjects and methods These polymorphisms were analyzed by polymerase chain reaction in 125 patients with obesity, 47 (T2DM) and 78 (Control Group). Results No significant difference was found on comparing the T2DM and Control Groups in respect to the genotypic frequencies of the polymorphisms - (II: 13.3% vs. 12.0%; ID: 37.8% vs. 37.3; DD: 48.9% vs. 50.7%; CC: 36.2% vs. 39.0%; CT: 46.8% vs. 49.3%; TT: 17.0% vs. 11.7%), and alleles (I: 32.2% vs. 30.7%; D: 67.8% vs. 69.3%; C: 59.6% vs. 63.6%; T: 40.4% vs. 36.4%) and their synergisms in the pathophysiology of T2DM. On analyzing the T2DM Group, there were no significant differences in the presence of complications. In this population of Brazilian obese patients, no correlation was found between the ACE and MTHFR polymorphisms in the development of T2DM. Conclusion Analyzing only the group with diabetes, there was also no relationship between these polymorphisms and comorbidities.


Subject(s)
Humans , Male , Female , Adolescent , Adult , Middle Aged , Aged , Young Adult , Polymorphism, Genetic/genetics , Peptidyl-Dipeptidase A/genetics , Methylenetetrahydrofolate Reductase (NADPH2)/genetics , Diabetes Mellitus, Type 2/enzymology , Obesity/complications , Brazil , Case-Control Studies , Polymerase Chain Reaction , Risk Factors , Mutagenesis, Insertional , Gene Deletion , Genetic Predisposition to Disease , Diabetes Mellitus, Type 2/complications , Diabetes Mellitus, Type 2/genetics , Genotype , Obesity/enzymology
7.
Laboratory Animal Research ; : 264-269, 2018.
Article in English | WPRIM | ID: wpr-718841

ABSTRACT

Cell cycle dysfunction can cause severe diseases, including neurodegenerative disease and cancer. Mutations in cyclin-dependent kinase inhibitors controlling the G1 phase of the cell cycle are prevalent in various cancers. Mice lacking the tumor suppressors p16(Ink4a) (Cdkn2a, cyclin-dependent kinase inhibitor 2a), p19(Arf) (an alternative reading frame product of Cdkn2a,), and p27(Kip1) (Cdkn1b, cyclin-dependent kinase inhibitor 1b) result in malignant progression of epithelial cancers, sarcomas, and melanomas, respectively. Here, we generated knockout mouse models for each of these three cyclin-dependent kinase inhibitors using engineered nucleases. The p16(Ink4a) and p19(Arf) knockout mice were generated via transcription activator-like effector nucleases (TALENs), and p27(Kip1) knockout mice via clustered regularly interspaced short palindromic repeats/CRISPR-associated nuclease 9 (CRISPR/Cas9). These gene editing technologies were targeted to the first exon of each gene, to induce frameshifts producing premature termination codons. Unlike preexisting embryonic stem cell-based knockout mice, our mouse models are free from selectable markers or other external gene insertions, permitting more precise study of cell cycle-related diseases without confounding influences of foreign DNA.


Subject(s)
Animals , Cell Cycle , Codon, Nonsense , Cyclin-Dependent Kinase Inhibitor p16 , DNA , Exons , G1 Phase , Genome , Melanoma , Mice , Mice, Knockout , Mutagenesis, Insertional , Neurodegenerative Diseases , Phosphotransferases , Reading Frames , Sarcoma
8.
Braz. j. microbiol ; 47(4): 785-792, Oct.-Dec. 2016. tab
Article in English | LILACS | ID: biblio-828193

ABSTRACT

Abstract Acinetobacter baumannii is widely recognized as an important pathogen associated with nosocomial infections. The treatment of these infections is often difficult due to the acquisition of resistance genes. A. baumannii presents a high genetic plasticity which allows the accumulation of these resistance determinants leading to multidrug resistance. It is highlighted the importance of the horizontal transfer of resistance genes, through mobile genetic elements and its relationship with increased incidence of multidrug resistant A. baumannii in hospitals. Considering that resistance to carbapenems is very important from the clinical and epidemiological point of view, the aim of this article is to present an overview of the current knowledge about genetic elements related to carbapenem resistance in A. baumannii such as integrons, transposons, resistance islands and insertion sequences.


Subject(s)
DNA, Bacterial , DNA Transposable Elements , Carbapenems/pharmacology , beta-Lactam Resistance , Acinetobacter baumannii/drug effects , Acinetobacter baumannii/genetics , Anti-Bacterial Agents/pharmacology , Mutagenesis, Insertional , Integrons , Genomic Islands
9.
IJRM-Iranian Journal of Reproductive Medicine. 2016; 14 (5): 323-328
in English | IMEMR | ID: emr-180249

ABSTRACT

Background: Spontaneous abortion is considered as the most complex problem during pregnancy. Thrombophilia is resumed as a cause of recurrent pregnancy loss [RPL]. Glycoprotein IIIa [GPIIIa] gene is involved in thrombosis and abortion. Angiotensin converting enzyme [ACE] converts angiotensin I to angiotensin II and is involved in thrombosis. The most common polymorphism in this gene is the insertion/deletion [I/D]


Objective: In this study, we analyzed the association between ACE I/D and GPIIIa c.98C >T polymorphisms in women with unexplained RPL from the north of Iran


Materials and Methods: Sample population consisted of 100 women with unexplained RPL and 100 controls. The ACE I/D and GPIIIa c.98C>T polymorphisms were genotyped by TETRA-ARMS PCR. The association between genotypes frequency and RPL were analyzed using ?P2P and exact fisher tests. Associated risk with double genotype combinations was also investigated by binary logistic regression


Results: There was significant association between ACE DD genotype and RPL [OR=2.04; 95% CI=0.94-4.44; p=0.036]. ACE D Allele was also significantly associated with the RPL [OR=1.59; 95% CI=1.05-2.41; p=0.013]. No significant association was observed between GPIIIa c.98C>T polymorphism and RPL


Conclusion: ACE I/D polymorphism may probably be a prognostic factor in female family members of women with the history of recurrent abortion


Subject(s)
Adult , Humans , Women , Peptidyl-Dipeptidase A/genetics , Integrin beta3/genetics , Mutagenesis, Insertional , Sequence Deletion , Genetic Association Studies , Polymorphism, Genetic , Case-Control Studies
10.
Chinese Journal of Biotechnology ; (12): 409-429, 2016.
Article in Chinese | WPRIM | ID: wpr-337455

ABSTRACT

Long terminal repeat (LTR) retrotransposons are mobile DNA sequences that ubiquitously exist in eukaryotic genomes. They replicate themselves in the genome by copy-paste mechanism with RNA as medium. In higher plants, many active LTR retrotransposons have been applied to analyze molecular marker technology, genetic tagging, insertion mutation and gene function. Here, we systematically review the characteristics of plant active LTR retrotransposons, including their structures, copy numbers and distributions. We further analyzed the gag (group-specific antigen) and pol (polymerase) sequence features of different plants active LTR retrotransposons and the distribution patterns of the cis-acting elements in LTR regions. The results show that autonomous active LTR retrotransposons must contain LTR regions and code Gag, Pr, Int, Rt, Rh proteins. Both LTR regions are highly homologous with each other and contain many cis-regulatory elements; RVT and RNase_H1_RT domain are essential for Rt and Rh protein respectively. These results provide the basis for subsequent identification of plant active LTR retrotransposons and their functional analysis.


Subject(s)
Genome, Plant , Mutagenesis, Insertional , Plants , Genetics , Retroelements , Terminal Repeat Sequences
11.
Article in Chinese | WPRIM | ID: wpr-345384

ABSTRACT

<p><b>OBJECTIVE</b>To analyze the clinical features and potential mutations of the SLC25A13 gene in a boy affected with neonatal intrahepatic cholestasis.</p><p><b>METHODS</b>Clinical data and peripheral venous blood sample of the child, and peripheral venous blood samples of both parents, were collected. All coding exons of the SLC25A13 gene were amplified with PCR and subjected to direct DNA sequencing.</p><p><b>RESULTS</b>The boy was found to be a compound heterozygote carrying c.851_854delGTAT and IVS16ins3kb mutations of the SLC25A13 gene, which were respectively inherited from his mother and father.</p><p><b>CONCLUSION</b>Based on its clinical and genetic features, the patient was diagnosed with neonatal intrahepatic cholestasis caused by citrin deficiency.</p>


Subject(s)
Base Sequence , Cholestasis, Intrahepatic , Genetics , Citrullinemia , DNA Mutational Analysis , Family Health , Female , Heterozygote , Humans , Infant , Infant, Newborn , Male , Mitochondrial Membrane Transport Proteins , Genetics , Mutagenesis, Insertional , Mutation , Sequence Deletion
12.
Braz. j. microbiol ; 46(2): 601-611, Apr-Jun/2015. tab, graf
Article in English | LILACS | ID: lil-749726

ABSTRACT

Deinococcus radiodurans (DR) is an extremophile that is well known for its resistance to radiation, oxidants and desiccation. The gene dr1790 of D. radiodurans was predicted to encode a yellow-related protein. The primary objective of the present study was to characterize the biological function of the DR1790 protein, which is a member of the ancient yellow/major royal jelly (MRJ) protein family, in prokaryotes. Fluorescence labeling demonstrated that the yellow-related protein encoded by dr1790 is a membrane protein. The deletion of the dr1790 gene decreased the cell growth rate and sensitivity to hydrogen peroxide and radiation and increased the membrane permeability of D. radiodurans. Transcript profiling by microarray and RT-PCR analyses of the dr1790 deletion mutant suggested that some genes that are involved in protein secretion and transport were strongly suppressed, while other genes that are involved in protein quality control, such as chaperones and proteases, were induced. In addition, the expression of genes with predicted functions that are involved in antioxidant systems, electron transport, and energy metabolism was significantly altered through the disruption of dr1790. Moreover, the results of proteomic analyses using 2-DE and MS also demonstrated that DR1790 contributed to D. radiodurans survival. Taken together, these results indicate that the DR1790 protein from the ancient yellow protein family plays a pleiotropic role in the survival of prokaryotic cells and contributes to the extraordinary resistance of D. radiodurans against oxidative and radiation stresses.


Subject(s)
Deinococcus/genetics , Genes, Bacterial , Genetic Pleiotropy , Mutagenesis, Insertional , Bacterial Proteins/genetics , Cell Membrane/physiology , Deinococcus/drug effects , Deinococcus/growth & development , Deinococcus/radiation effects , Gene Deletion , Gene Expression Profiling , Genetic Complementation Test , Hydrogen Peroxide/toxicity , Microarray Analysis , Membrane Proteins/genetics , Microbial Viability/drug effects , Microbial Viability/radiation effects , Permeability , Radiation, Ionizing , Real-Time Polymerase Chain Reaction
13.
Indian J Biochem Biophys ; 2015 Apr; 52 (2): 209-212
Article in English | IMSEAR | ID: sea-158225

ABSTRACT

Diabetic nephropathy (DN) is a major cause of morbidity and mortality in diabetes. Vascular endothelial growth factor (VEGF) is a potent multi-functional cytokine which plays a key role in the pathogenesis of DN. In this study, we evaluated the possible association of the VEGF gene (I/D) polymorphisms with DN in type 2 diabetes patients in West Indian population. Genotyping (I/D) of the VEGF gene polymorphism was done by the polymerase chain reaction. A total of 103 patients with type 2 diabetes, 102 patients with DN, 108 patients with non-diabetic nephropathy and 143 healthy controls were genotyped. The frequency of VEGF genotype distribution and biochemical parameters like creatinine and HbA1c were compared in diabetic, diabetic nephropathy, non diabetic nephropathy and control groups. We found significant difference in creatinine level in DN and NDN groups on comparison with control group. Our study suggests that I/D polymorphism in the promoter region of the VEGF gene is not associated with DN in type 2 diabetes patients, but might have a role in development of non-diabetic nephropathy.


Subject(s)
Diabetes Mellitus, Type 2/epidemiology , Diabetes Mellitus, Type 2/genetics , Diabetic Nephropathies/epidemiology , Diabetic Nephropathies/genetics , Gene Deletion , Genotyping Techniques/methods , Humans , India , Mutagenesis, Insertional/genetics , Polymorphism, Genetic/genetics , Vascular Endothelial Growth Factor A/genetics
14.
Chinese Journal of Biotechnology ; (12): 1741-1752, 2015.
Article in Chinese | WPRIM | ID: wpr-337461

ABSTRACT

Insertional mutagenesis is a widely used method to determine the function(s) of a gene. To study the function(s) of the gene nsdAmgh in Streptomyces roseoflavus, a homologous recombination vector pSRNA2500 was structured in this paper. The recombination donor vector was then transformed into Strempomyces roseoflavus strain Men-myco-93-63 by conjugative transfer. The transformants were subjected to selection under the pressure of high temperature and appropriate antibiotics. As a result, several disrupted mutants of nsdAmgh gene, with a phenotype of Am(s)Km(r), were isolated and verified using PCR and Dot-blotting and Southern blotting hybridization methods. Functional analysis showed that the disrupted mutants of nsdAmgh had a two-fold higher inhibition against Verticillium dahlia Kleb than that of the wild strain Men-myco-93-63, which all will provide a new study route for future research about positive and negative regulator in Men-myco-93-63.


Subject(s)
Genes, Bacterial , Genetic Vectors , Mutagenesis, Insertional , Polymerase Chain Reaction , Streptomyces , Genetics
15.
Chinese Journal of Hepatology ; (12): 13-16, 2015.
Article in Chinese | WPRIM | ID: wpr-337057

ABSTRACT

<p><b>OBJECTIVE</b>To explore the clinical features and gene mutation profiles of patients with chronic hepatitis B (CHB) and Gilbert's syndrome.</p><p><b>METHODS</b>Thirty-three patients with CHB and Gilbert's syndrome were enrolled in the study. Serum markers of liver function and histological features of disease-related liver injury were assessed by standard methods. Gene mutations were detected by PCR and direct DNA sequencing.Statistical analysis was carried out with the chi-square and t tests.</p><p><b>RESULTS</b>Sequencing of the Gilbert syndrome-associated gene, UGT 1A 1, revealed mutations in the upstream promoter phenobarbital-responsive element module (PBREM) (-3279 mutation, 23 cases), in the promoter TATA box (a TA insertion mutation, 21 cases), and in the coding region of exon 1 (a GGA-AGA Gly71Arg mutation, 18 cases); there was no statistical difference found for any of the three mutations among this patient population (x2 =1.640, P more than 0.05).</p><p><b>CONCLUSION</b>The traditional methods of diagnosis for patients with CHB and Gilbert's syndrome remain a technical challenge in the clinic, and gene detection may represent a more favorable method for diagnosing this patient population.</p>


Subject(s)
Base Sequence , Exons , Gilbert Disease , Glucuronosyltransferase , Hepatitis B, Chronic , Humans , Mutagenesis, Insertional , Mutation , Polymerase Chain Reaction , Promoter Regions, Genetic , TATA Box
16.
Chinese Journal of Biotechnology ; (12): 1162-1174, 2015.
Article in Chinese | WPRIM | ID: wpr-240567

ABSTRACT

Genetic modification technology is a new molecular tool for targeted genome modification. It includes zinc finger nucleases (ZFN) technology, transcription activator-like effector nucleases (TALEN) technology and clustered regularly interspaced short palindromic repeat (CRISPR)-associated (Cas) (CRISPR-Cas) nucleases technology. All of these nucleases create DNA double-strand breaks (DSB) at chromosomal targeted sites and induce cell endogenous mechanisms that are primarily repaired by the non-homologous end joining (NHEJ) or homologous recombination (HR) pathway, resulting in targeted endogenous gene knock-out or exogenous gene insertion. In recent years, genetic modification technologies have been successfully applied to bacteria, yeast, human cells, fruit fly, zebra fish, mouse, rat, livestock, cynomolgus monkey, Arabidopsis, rice, tobacco, maize, sorghum, wheat, barley and other organisms, showing its enormous advantage in gene editing field. Especially, the newly developed CRISPR-Cas9 system arose more attention because of its low cost, high effectiveness, simplicity and easiness. We reviewed the principles and the latest research progress of these three technologies, as well as prospect of future research and applications.


Subject(s)
Animals , CRISPR-Cas Systems , DNA Breaks, Double-Stranded , Endonucleases , Genetic Engineering , Methods , Humans , Mutagenesis, Insertional , Mutagenesis, Site-Directed , Plants , Zinc Fingers
17.
Article in Chinese | WPRIM | ID: wpr-239493

ABSTRACT

<p><b>OBJECTIVE</b>To explore the molecular basis for an individual with rare p phenotype in the P1Pk blood group system.</p><p><b>METHODS</b>Erythrocyte blood group antigens and antibodies in serum were identified in the proband and five family members with a serological method. Coding regions and flanking untranslated regions of the α1,4-galactosyltransferase gene (A4GALT) encoding P1Pk antigens were amplified with polymerase chain reaction and directly sequenced. The haplotypes of A4GALT in the parents of the proband were also analyzed by cloning sequencing.</p><p><b>RESULTS</b>The proband was found with a rare p phenotype with anti-Tja antibody in his serum by serological method. The other family members all had a common P2 phenotype. The results of DNA sequencing showed that a cytosine was inserted at nucleotide position 1026 to 1029 (1026_1029insC) of both alleles of the A4GALT gene in the proband. The mutation has caused a reading frame shift and formed a mutant protein by extending 92 amino acid residues. The other family members were either heterozygous for the insertion or of the wild type at above position.</p><p><b>CONCLUSION</b>The 1026_1029insC mutation of the A4GALT gene is probably responsible for the p phenotype identified for the first time in Chinese population. The individual with the p phenotype possesses anti-Tja antibody.</p>


Subject(s)
ABO Blood-Group System , Genetics , Adult , Alleles , Asian Continental Ancestry Group , Genetics , Base Sequence , Female , Frameshift Mutation , Galactosyltransferases , Genetics , Humans , Male , Molecular Sequence Data , Mutagenesis, Insertional , Pedigree , Phenotype , Young Adult
18.
Article in Korean | WPRIM | ID: wpr-217692

ABSTRACT

Genome editing is a useful research tool essentially applicable to gene therapy in the field of biotechnology, pharmaceutics and medicine. Scientists have developed three types of programmable nucleases for genome editing, and these include: Zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeat (CRISPR)-Cas (CRISPR-associated) system particularly derived from bacterial adaptive immune system. Programmable nucleaseses occur double strand breaks (DSBs) on target strand, and a repair mechanism of DSBs introduces either non-homologous end joining (NHEJ) or homology directed repair (HDR), where the pathway is determined by presence of donor DNA template. In this sense, we can generate gene insertion, gene correction, point mutagenesis and chromosomal translocations via endogenous repair mechanism. However, these nucleases exhibit several discrepancies in the aspects of their compositions, targetable sites, efficiency and other characteristics. Here, we discuss on various characteristics of three programmable nucleases and potential outcomes of DSBs. Acknowledging the distinctions among these programmable nucleases will help scientists to select appropriate tools in genome engineering.


Subject(s)
Biotechnology , Clustered Regularly Interspaced Short Palindromic Repeats , Deoxyribonuclease I , DNA , Genetic Engineering , Genetic Therapy , Genome , Humans , Immune System , Mutagenesis , Mutagenesis, Insertional , Tissue Donors , Translocation, Genetic
19.
Article in English | WPRIM | ID: wpr-115861

ABSTRACT

Hypoparathyroidism, sensorineural deafness, and renal dysgenesis syndrome is an autosomal dominant disease caused by mutations in the GATA3 gene on chromosome 10p15. We identified a patient diagnosed with hypoparathyroidism who also had a family history of hypoparathyroidism and sensorineural deafness, present in the father. The patient was subsequently diagnosed and found to be a heterozygote for an insertion mutation c.255_256ins4 (GTGC) in exon 2 of GATA3. His father was also confirmed to have the same mutation in GATA3.


Subject(s)
Deafness , Exons , Fathers , Hearing Loss, Sensorineural , Heterozygote , Humans , Hypoparathyroidism , Mutagenesis, Insertional
20.
Mem. Inst. Oswaldo Cruz ; 109(8): 1081-1085, 12/2014. graf
Article in English | LILACS | ID: lil-732602

ABSTRACT

We present here three expression plasmids for Trypanosoma cruzi adapted to the Gateway® recombination cloning system. Two of these plasmids were designed to express trypanosomal proteins fused to a double tag for tandem affinity purification (TAPtag). The TAPtag and Gateway® cassette were introduced into an episomal (pTEX) and an integrative (pTREX) plasmid. Both plasmids were assayed by introducing green fluorescent protein (GFP) by recombination and the integrity of the double-tagged protein was determined by western blotting and immunofluorescence microscopy. The third Gateway adapted vector assayed was the inducible pTcINDEX. When tested with GFP, pTcINDEX-GW showed a good response to tetracycline, being less leaky than its precursor (pTcINDEX).


Subject(s)
Gene Expression/genetics , Genetic Vectors/genetics , Plasmids , Restriction Mapping/methods , Trypanosoma cruzi/genetics , Blotting, Western , Expressed Sequence Tags/metabolism , Green Fluorescent Proteins , Life Cycle Stages/genetics , Mutagenesis, Insertional , Tetracycline/pharmacology , Trypanosoma cruzi/drug effects
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