• MeSH
• CRISPR-Cas systémy MeSH
• genetická terapie * metody   MeSH
• lidé MeSH
• nádory * terapie   MeSH
• TALENs terapeutické užití   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• přehledy MeSH

Approximately 35 % of the mouse genes are indispensable for life, thus, global knock-out (KO) of those genes may result in embryonic or early postnatal lethality due to developmental abnormalities. Several KO mouse lines are valuable human disease models, but viable homozygous mutant mice are frequently required to mirror most symptoms of a human disease. The site-specific gene editing systems, the transcription activator-like effector nucleases (TALENs), Zinc-finger nucleases (ZFNs) and the clustered regularly interspaced short palindrome repeat-associated Cas9 nuclease (CRISPR/Cas9) made the generation of KO mice more efficient than before, but the homozygous lethality is still an undesired side-effect in case of many genes. The literature search was conducted using PubMed and Web of Science databases until June 30th, 2020. The following terms were combined to find relevant studies: "lethality", "mice", "knock-out", "deficient", "embryonic", "perinatal", "rescue". Additional manual search was also performed to find the related human diseases in the Online Mendelian Inheritance in Man (OMIM) database and to check the citations of the selected studies for rescuing methods. In this review, the possible solutions for rescuing human disease-relevant homozygous KO mice lethal phenotypes were summarized.

• MeSH
• CRISPR-Cas systémy genetika   MeSH
• editace genu metody   MeSH
• fenotyp MeSH
• myši knockoutované MeSH
• myši MeSH
• nukleasy s motivem zinkových prstů genetika   MeSH
• TALENs genetika   MeSH
• ztráta embrya genetika   prevence a kontrola   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH

Reporter gene mouse lines are routinely used for studies related to functional genomics, proteomics, cell biology or cell-based drug screenings, and represent a crucial platform for in vivo research. In the generation of knock-in reporter lines, new gene targeting methods provide several advantages over the standard transgenic techniques. First of all, specific targeting of the genome allows expression of the reporter gene under controlled conditions, whether in a specific locus in the genome or in a "safe harbor" locus. Historically, the ROSA26 locus is used for gene knock-in strategies by homologous recombination in mouse embryonic stem cells. The other preferred place for integration of the reporter transgene in the mouse genome is the endogenous promoter of a target gene. In this study, we employed TALENs to generate a reporter fusion protein expressed from its native promoter. For monitoring DNA damage response, we generated a mouse line expressing a mCitrine-tagged version of the FANCD2 protein, involved in DNA damage response and repair, and the Fanconi anemia (FA) pathway. This model could be a valuable tool for in vivo investigation of DNA damage.

• MeSH
• bakteriální proteiny MeSH
• luminescentní proteiny MeSH
• modely u zvířat * MeSH
• myši inbrední C57BL MeSH
• myši MeSH
• oprava DNA MeSH
• poškození DNA MeSH
• protein FANCD2 genetika   MeSH
• rekombinantní fúzní proteiny * MeSH
• reportérové geny * MeSH
• TALENs MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Silene latifolia serves as a model species to study dioecy, the evolution of sex chromosomes, dosage compensation and sex-determination systems in plants. Currently, no protocol for genetic transformation is available for this species, mainly because S. latifolia is considered recalcitrant to in vitro regeneration and infection with Agrobacterium tumefaciens. Using cytokinins and their synthetic derivatives, we markedly improved the efficiency of regeneration. Several agrobacterial strains were tested for their ability to deliver DNA into S. latifolia tissues leading to transient and stable expression of the GUS reporter. The use of Agrobacterium rhizogenes strains resulted in the highest transformation efficiency (up to 4.7% of stable transformants) in hairy root cultures. Phenotypic and genotypic analyses of the T1 generation suggested that the majority of transformation events contain a small number of independent T-DNA insertions and the transgenes are transmitted to the progeny in a Mendelian pattern of inheritance. In short, we report an efficient and reproducible protocol for leaf disc transformation and subsequent plant regeneration in S. latifolia, based on the unique combination of infection with A. rhizogenes and plant regeneration from hairy root cultures using synthetic cytokinins. A protocol for the transient transformation of S.latifolia protoplasts was also developed and applied to demonstrate the possibility of targeted mutagenesis of the sex linked gene SlAP3 by TALENs and CRISPR/Cas9.

• MeSH
• Agrobacterium genetika   MeSH
• chromozomy rostlin genetika   MeSH
• CRISPR-Cas systémy MeSH
• DNA bakterií genetika   MeSH
• exprese genu MeSH
• genetické techniky MeSH
• geneticky modifikované rostliny MeSH
• modely genetické MeSH
• molekulární evoluce MeSH
• regenerace genetika   MeSH
• reportérové geny MeSH
• Silene genetika   mikrobiologie   fyziologie   MeSH
• TALENs MeSH
• transformace genetická * MeSH
• Publikační typ
• časopisecké články MeSH

• MeSH
• amplifikace genu genetika   MeSH
• inzerční mutageneze MeSH
• mutageneze * genetika   MeSH
• poškození DNA genetika   MeSH
• rekombinantní fúzní proteiny MeSH
• TALENs MeSH
• transpozibilní elementy DNA genetika   MeSH
• zlomy DNA MeSH

We report a comprehensive toolkit that enables targeted, specific modification of monocot and dicot genomes using a variety of genome engineering approaches. Our reagents, based on transcription activator-like effector nucleases (TALENs) and the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system, are systematized for fast, modular cloning and accommodate diverse regulatory sequences to drive reagent expression. Vectors are optimized to create either single or multiple gene knockouts and large chromosomal deletions. Moreover, integration of geminivirus-based vectors enables precise gene editing through homologous recombination. Regulation of transcription is also possible. A Web-based tool streamlines vector selection and construction. One advantage of our platform is the use of the Csy-type (CRISPR system yersinia) ribonuclease 4 (Csy4) and tRNA processing enzymes to simultaneously express multiple guide RNAs (gRNAs). For example, we demonstrate targeted deletions in up to six genes by expressing 12 gRNAs from a single transcript. Csy4 and tRNA expression systems are almost twice as effective in inducing mutations as gRNAs expressed from individual RNA polymerase III promoters. Mutagenesis can be further enhanced 2.5-fold by incorporating the Trex2 exonuclease. Finally, we demonstrate that Cas9 nickases induce gene targeting at frequencies comparable to native Cas9 when they are delivered on geminivirus replicons. The reagents have been successfully validated in tomato (Solanum lycopersicum), tobacco (Nicotiana tabacum), Medicago truncatula, wheat (Triticum aestivum), and barley (Hordeum vulgare).

• MeSH
• genetické inženýrství metody   MeSH
• geneticky modifikované rostliny genetika   MeSH
• ječmen (rod) genetika   MeSH
• pšenice genetika   MeSH
• RNA rostlin genetika   MeSH
• rostlinné proteiny genetika   MeSH
• sekvence CRISPR genetika   MeSH
• Solanum lycopersicum genetika   MeSH
• TALENs genetika   MeSH
• Publikační typ
• časopisecké články MeSH

Recently, it has been found that spontaneous mutation Lx (polydactyly-luxate syndrome) in the rat is determined by deletion of a conserved intronic sequence of the Plzf (Promyelocytic leukemia zinc finger protein) gene. In addition, Plzf is a prominent candidate gene for quantitative trait loci (QTLs) associated with cardiac hypertrophy and fibrosis in the spontaneously hypertensive rat (SHR). In the current study, we tested the effects of Plzf gene targeting in the SHR using TALENs (transcription activator-like effector nucleases). SHR ova were microinjected with constructs pTAL438/439 coding for a sequence-specific endonuclease that binds to target sequence in the first coding exon of the Plzf gene. Out of 43 animals born after microinjection, we detected a single male founder. Sequence analysis revealed a deletion of G that resulted in frame shift mutation starting in codon 31 and causing a premature stop codon at position of amino acid 58. The Plzftm1Ipcv allele is semi-lethal since approximately 95% of newborn homozygous animals died perinatally. All homozygous animals exhibited manifestations of a caudal regression syndrome including tail anomalies and serious size reduction and deformities of long bones, and oligo- or polydactyly on the hindlimbs. The heterozygous animals only exhibited the tail anomalies. Impaired development of the urinary tract was also revealed: one homozygous and one heterozygous rat exhibited a vesico-ureteric reflux with enormous dilatation of ureters and renal pelvis. In the homozygote, this was combined with a hypoplastic kidney. These results provide evidence for the important role of Plzf gene during development of the caudal part of a body-column vertebrae, hindlimbs and urinary system in the rat.

• MeSH
• alely MeSH
• DNA vazebné proteiny nedostatek   genetika   metabolismus   MeSH
• exony MeSH
• genotyp MeSH
• genový targeting MeSH
• heterozygot MeSH
• homozygot MeSH
• krysa rodu rattus MeSH
• lokus kvantitativního znaku MeSH
• mnohočetné abnormality genetika   patologie   veterinární   MeSH
• ocas abnormality   MeSH
• polydaktylie genetika   patologie   veterinární   MeSH
• posunová mutace MeSH
• potkani inbrední SHR MeSH
• sekvence aminokyselin MeSH
• sekvence nukleotidů MeSH
• TALENs genetika   metabolismus   MeSH
• vazba proteinů MeSH
• zvířata MeSH
• Check Tag
• krysa rodu rattus MeSH
• mužské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

Engineered nucleases are able to introduce double stranded breaks at desired genomic locations. The breaks can be repaired by an error-prone non-homologous end joining (NHEJ) mechanism, or the repair process can be exploited to introduce precise DNA modifications by homology-directed repair (HDR) when provided with a suitable donor template. We designed a series of DNA donors including long dsDNA plasmids as well as short ssDNA oligonucleotides and compared the effectiveness of their utilization during gene targeting with highly efficient transcription activator-like effector nucleases (TALENs). While the use of long dsDNA donors for the incorporation of larger DNA fragments in Bombyx is still a problem, short single-stranded oligodeoxynucleotides (ssODNs) are incorporated quite efficiently. We show that appropriately designed ssODNs were integrated into germ cells in up to 79% of microinjected individuals and describe in more detail the conditions for the precise genome editing of Bombyx genes. We specify the donor sequence requirements that affected knock-in efficiency, and demonstrate the successful applications of this method of sequence deletion, insertion and replacement in the Bombyx genome.

• MeSH
• bourec genetika   MeSH
• DNA genetika   metabolismus   MeSH
• editace genu metody   MeSH
• jednovláknová DNA genetika   metabolismus   MeSH
• TALENs genetika   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

Jedním z největších cílů genové terapie je dosáhnout výměny defektního genu, nebo jeho části, za funkční gen ve své přirozené chromosomální lokalizaci a tím se vyvarovat začlenění celého terapeutického genu s případnými cizími či regulačními sekvencemi. Navržený projekt bude zavádět a ověřovat novou technologii, která spočívá ve speciální cílené terapii mutací způsobujících monogenetické choroby. Tato technologie je založena na využití transcription activator–like effector nukleáz (TALEN), které pomohou vkládat korigované (normálně funkční) sekvence na místo poškozené DNA. K ověření genově-terapeutických možností byly vybrány dvě onemocnění: Diamond–Blackfan anemie (DBA) a hemofilie A. Dílčími cíly projektu jsou: vytvoření nových myších modelů zmíněných chorob pomocí TALEN-technologie tak, aby lépe odpovídaly lidským mutacím, korekce mutací pomocí genové terapie, vytvoření sady specifických TALE-nukleáz ke korekci mutací u lidí a jejich validace v buňkách získaných od pacientů.; One of the biggest goals of the gene therapy is to replace the defective gene or its part by the fully functional gene at the natural chromosomal localization. This would avoid the insertion of the whole therapeutic gene including foreign or regulation sequences. The project will establish and evaluate the new technology based on special therapeutical targeting of mutations causing monogenetic diseases. This technology relies on transcription activator-like effector nucleases (TALENs) that will help to insert corrected sequences to the site of damaged DNA. To confirm gene-therapeutical possibilities we selected: Diamond-Blackfan anemia (DBA) and hemophilia A. Particular goals are: the generation of new mouse models of the selected diseases using TALEN-technology to resemble better the human mutations; the correction of mutations using gene therapy; the establishment of the set of specific TALE-nucleases for the repair of human mutations; their validation using cells obtained from patients.

• MeSH
• Diamondova-Blackfanova anemie MeSH
• faktor VIII MeSH
• fenotyp MeSH
• genetická terapie MeSH
• hemofilie A MeSH
• modely nemocí na zvířatech MeSH
• myši MeSH
• ribozomální proteiny MeSH
• TALENs MeSH
• Check Tag
• myši MeSH

• Konspekt
• Biochemie. Molekulární biologie. Biofyzika
• NLK Obory
• molekulární biologie, molekulární medicína
• hematologie a transfuzní lékařství
• NLK Publikační typ
• závěrečné zprávy o řešení grantu IGA MZ ČR