Knockout mice created by talen-mediated gene targeting pdf

Electroporation of ascpf1rnp at the zygote stage is an. Knockout mice created by talenmediated gene targeting young hoon sung, injeoung baek, duk hyoung kim, jisun jeon, jaehoon lee, kyunghee lee, daewon jeong, jinsoo kim, and hanwoong lee presented by jessica april 3rd, 20 20. Talen mediated gene targeting in avian primordial germ cells is therefore an efficient process. Doublestrand dna breaks dramatically enhance hr 10. Genomic dna was isolated from cells harvested 72 hr after transient transfection and screened for the presence of sitespecific gene modification by pcr amplification of regions surrounding the target sites as well as t7en1 cleavage assay figure 1b. Transcription activatorlike effector nucleases talens are a new type of engineered nuclease that is very effective for directed gene disruption in any genome sequence. We investigated the generation of mice with genetic knockout ko of the g proteincoupled receptor kinase grk 5 gene by microinjection of talen mrna. Sitespecific knockin is another advantage of nucleasemediated gene targeting in large animals. Using talens as sequencespecific nucleases to induce targeted doublestrand breaks, the mouse genome can be directly modified in zygotes in a single step without the need for embryonic stem cells. Ascpf1mediated lif ko mice showed postnatal growth retardation and implantation failure, both of which are major phenotypes of lif ko mice generated by conventional gene targeting. In this study, we report the detailed phenotype of eif2s3y knockout mice generated using talenmediated gene disruption.

Gene knockout ko mice are powerful tools for studying biological science and genetic diseases of humans skarnes et al. Grk5knockout mice generated by talenmediated gene targeting article pdf available in animal biotechnology 274. Transcription activatorlike effector nucleases talen were designed against. The value of knockout mouse models a detailed analysis. A knockout mouse is a mouse in which an existing gene has been inactivated, or knocked out, by deleting portions of the gene s dna sequence to make it nonfunctional, or by replacing the gene with an altered dna sequence get a quote. Recent advances in gene editing technology have introduced the potential for application of mutagenesis approaches in nonhuman primates to model human development and disease. Pdf transcription activatorlike effector nucleases talens are programmable. Carlson df, tan w, lillico sg, stverakova d, proudfoot c, christian m, voytas df, long cr, whitelaw cb, fahrenkrug sc proc natl acad sci u s a. Phenotypic analysis of genespecific knockout mice has transformed our understanding of in vivo gene functions. Transcription activatorlike effector nucleases talens are representative nextgeneration platforms for customized genomic editing in transgenic animals, as. Conventional embryonic stem es cell mediated gene targeting requires at least 6 months to obtain homozygous mutant mice.

As talenmediated knockout chickens are genetically modified but nontransgenic, without genomic integration. We investigated the generation of mice with genetic knockout ko of the g proteincoupled receptor kinase grk 5 gene by microinjection of. Efficient talenmediated gene knockout in livestock. Generation of knockout mice, however, remains a timeconsuming and expensive process. The connection between knockout mice and human genetics has to do with the fact that 99% of the roughly 20,00025,000 genes in the human body actually have a mouse dna counterpart. Talen mediated endogenous gene disruption using oocytes from female mice at different weeks of age. This lecture about transgenic animals explains the mechanism of gene knockout mice production. The feasibility of talenmediated gene disruption has been confirmed in creating gene knockout mice and rats recently. Knockout mice created by talen mediated gene targeting. The talenmediated method is suitable for gene disruption of such repeat embedded genes and can be used to more quickly obtain ko mice since it. Oct 23, 2012 transcription activatorlike effector nucleases talens are programmable nucleases that join foki endonuclease with the modular dnabinding domain of tales. The monkey mecp2 gene contains 4 exons, similar to that of humans and mice figure 1a, with the methylcpg binding domain mbd encoded by both exon 3 and exon 4 nan et al.

Dec 01, 20 read tolllike receptor 4 tlr4 knockout rats produced by transcriptional activatorlike effector nuclease talen mediated gene inactivation, alcohol on deepdyve, the largest online rental service for scholarly research with thousands of academic publications available at your fingertips. Claudin4 knockout by talenmediated gene targeting in mdck cells. To our knowledge, this is first report of targeted mutagenesis in monkeys with talen technology for modeling of human disease. Efficient talen mediated gene knockout in livestock. Gene engineering for generating targeted mouse mutants is a key technology for biomedical research. In this study, we aimed to test whether targeted mstn editing, mediated by transcription activatorlike effector nucleases talens, is a viable approach to create myostatinmodified goats capra hircus. Schematic representation of the genetic engineering methods used for generating targeted knockout rats. One of the costeffective way is routinely used to generate geneticallymodified small animals is directed injection of the gene targeting nucleases into zygote, including rat, mice, and rabbit. Transcription activatorlike tal effector nucleases talens are highly effective in inducing mutations at specific genomic loci, and consequently talenmediated mutagenesis in zygotes is a potential. In this study, we accomplished targeted knockout of the ovalbumin ov gene in the chicken. The convenience of this technique means that t alen mediated. Highly efficient targeted mutagenesis in onecell mouse embryos. However, many of the resulted founder animals were chimeric ones with multiple mutations through the embryo injection 17.

Gene editing nuclease and its application in tilapia. Timeline and generation of mutant mice by single plasmid injection. Knockout rat models mimicking human atherosclerosis created by cpf1mediated gene targeting. We amputated all four limbs of each animal to examine the effect of tsp. Sung yh, baek ij, kim dh, jeon j, lee j, lee k, jeong d, kim js, lee hw.

In ddx4 knockout chickens, pgcs are initially formed but are lost during meiosis in the developing ovary, leading to adult female sterility. Although zincfinger nucleases enable a variety of genome modifications, their application to genetic engineering of livestock has been slowed by technical limitations of embryoinjection, culture of primary cells, and difficulty in. Gene editing in model organisms has provided critical insights into brain development and diseases. Anephrogenic phenotype induced by sall1 gene knockout in. Here we report successful talen mediated mutagenesis of an xlinked, rett. Es cell gene targeting technique to be defective in a particular gene product provide an attractive model system in which to. Talenmediated gene targeting in avian pgcs is therefore an efficient process.

The most commonly used method consists of three major steps. Aspects of the technology for generating knockout mice, and the mice themselves have been patented in many countries by private companies. Gene targeting by taleninduced homologous recombination. Highefficiency and heritable gene targeting in mouse by. Capecchi, martin evans, and oliver smithies in 1989, for which they were awarded the 2007 nobel prize in physiology or medicine. But, gene targeting in human pluripotent stem cells including es and ips cells has been very difficult historically. Targeted gene deletion of mirnas in mice by talen system. Knockout mice created by talen mediated gene targeting to the editor. Claudin4 knockout by talenmediated gene targeting in. Transcriptomics of gabra4 knockout mice reveals common nmdar pathways underlying autism, memory, and epilepsy cuixia fan, yue gao, guanmei liang, lang huang, jing wang.

We have created a strain of mice that lacks the y subtype of pkc to evaluate. Crisprcas9based genome editing in mice by single plasmid. Efficient talenmediated gene knockout in livestock pnas. Screening methods to identify talenmediated knockout mice. Pdf efficient talenmediated gene knockout in livestock. Genome editing in mice using tale nucleases request pdf. Transcriptomics of gabra4 knockout mice reveals common nmdar pathways underlying autism, memory, and epilepsy.

Genome editing in mice using tale nucleases springerlink. Pdf knockout mice created by talenmediated gene targeting. In many model systems, this approach has been invaluable for discovering the function of ge. Applying the method to knock in a series of cry1 mutants into cry1. Recently, a taleninduced y chromosomal gene modification in embryonic stem cells and oocytes was successfully utilized to generate knockout mice 14,15. The apoerat was created by talen mediated gene targeting in the genetic background of sprague dawley rat. Generation of genemodified cynomolgus monkey via cas9rna. Transcriptomics of gabra4 knockout mice reveals common nmdar pathways underlying autism, memory, and epilepsy cuixia fan, yue gao, guanmei liang, lang huang, jing wang, xiaoxue yang. Efficient talenmediated myostatin gene editing in goats. A read is counted each time someone views a publication summary such as the title, abstract, and list of authors, clicks on a figure, or views or downloads the fulltext. Efficient biallelic gene knockout and sitespecific knock. A, left and right target sequences of the mstn gene for d10a. Developing genetically engineered mouse models using. In ddx4 knockout chickens, pgcs are initially formed but are lost during meiosis in the developing ovary leading to adult female sterility.

All the aforementioned initiatives have the possibility to contribute to remarkable new treatments that could save many lives. Generating mouse models for biomedical research disease. The type ii bacterial crisprcas system is a novel genomeengineering technology with the ease of multiplexed gene targeting. Particularly exciting in livestock was the report of zfnmediated, biallelic ko of the porcine ggta1 gene using commercial zfn reagents 9, wherein biallelicnull cells could be enriched by facs for the absence of a ggta1 dependent surface epitope. Phenotypic analysis of gene specific knockout mice has transformed our understanding of in vivo gene functions. These techniques target the detailed study of gene activity. Crisprcas9 technique was able to efficiently modify alk1 and create the target mice.

Here, we report the generation of a cynomolgus monkey macaca fascicularis carrying mecp2 mutations using transcription activatorlike effector nucleases talensmediated gene targeting. Insights derived from ace knockout mice justin cole, dilek ertoy, kenneth e bernstein keywords. Efficient talenmediated myostatin gene editing in goats pdf. Genetargeted mutant animals, such as knockout or knockin mice, have dramatically improved our. Our results extended the application of stateoftheart talen technology from experimental animals to farm animals. Onestep generation of mice carrying mutations in multiple. However, the generation of mice with specific mutations is labor intensive and involves gene targeting by homologous recombination in es cells, the production of chimeric mice, and, after germline transmission of the targeted es cells, the interbreeding of heterozygous mice to produce the homozygous experimental animals, a process that may take. We discuss how conventional gene targeting by homologous. Talenmediated gene mutagenesis in rhesus and cynomolgus. Knockout rat models mimicking human atherosclerosis. G proteincoupled receptor kinase 5 grk5 is one of the seven grk family members whose primary function is to desensitize g proteincoupled receptors gpcrs. Consequently, observing the characteristics of knockout mice gives researchers information that can be used to better understand how a similar gene may cause or contribute to disease in humans.

Jul 27, 2016 myostatin mstn encodes a negative regulator of skeletal muscle mass that might have applications for promoting muscle growth in livestock. Department of biochemistry, college of life science. Dec 27, 20 gene targeting technologies with zinc finger nucleases zfns, transcription activator. Targeted gene knockout in chickens mediated by talens pnas. Talenmediated sitespecific insertion using ssodn with a hindiii site was successfully introduced to the somatic cell genome, however, a base substitution just before the hindiii site was identified. Apolipoprotein edeficient rats develop atherosclerotic. Knockout rat models mimicking human atherosclerosis created. In this study, we used genomeediting tools to create and analyze targeted somatic mutations in murine models of liver cancer. Production of sry knockout mouse using talen via oocyte. To explore the regionspecific genome functions in vivo, generation of gene targeting animals is a powerful strategy.

Grk5knockout mice generated by talenmediated gene targeting. After injecting talens mrna into monkey zygotes achieved by in vitro. Importantly, the crisprinduced tumors had features. T1 knockout mice created by talen mediated gene targeting. Application of oocyte cryopreservation technology in talen. To target these genes in the mouse genome, we designed and synthesized highly active. Crisprcas9 mediated gene modification utilizes doublestrand breaks dsbs and the host cells own dna repair systems. Request pdf genome editing in mice using tale nucleases gene engineering for generating targeted mouse mutants is a key technology for biomedical research. Cpf1 mediated generation of apoe andor ldlr knockout rats. Targeted gene knockout by editing specific loci in genome has revolutionized the field of functional genomics.

Recently, two new genetargeting technologies have been developed. These mice are produced by either inactivating a specific gene thought to play a key role in the development of these diseases, or inserting a new dna sequence in the case of knockin mouse models. Generation of conditional acvrl1 knockout mice by crispr. The efficiency of all sgrnas was first tested by cotransfection with cas9 into the cos7 cell line derived from african green monkey kidney. Jan 09, 20 read knockout mice created by talen mediated gene targeting, nature biotechnology on deepdyve, the largest online rental service for scholarly research with thousands of academic publications available at your fingertips. We further tested talen mediated endogenous gene targeting using fresh or freezethawed oocytes of c57bl6n mice for the general production of genomeedited mice using cryopreserved oocytes fig. Targeted gene knockout in chickens mediated by talens. After examining the efficient conditions, we injected two grnas targeting different loci on the same chromosome into mouse zygotes at once and addressed whether largescale genomemodified mice could be produced using cas9grna system in a single step procedure. Talenmediated somatic mutagenesis in murine models of cancer. Sep 02, 2014 in this study, in combination with chicken primordial germ cell line with germline transmission capacity, we generated the ovalbumin gene knockout chickens by talen mediated gene targeting. Sixto eightweekold male rats were used in the experiments n 10 in each group. Pdf grk5knockout mice generated by talenmediated gene. The primary goal of this study was to establish an atherosclerotic rat model. Knockout mice created by talenmediated gene targeting generation of knockout mice, however, remains a timeconsuming and expensive process.

To examine whether cpf1 is suitable for gene targeting in rats, we used the cpf1 ortholog from lachnospiraceae bacterium n d2006 lbcpf1 and generated a crispr rna crrna targeting exon 3 of the rat apolipoprotein e apoe gene apoecrrna, which is common among the rat apoe transcript variants fig. Although thousands of genes have been disrupted in mice by conventional gene targeting 6, investigators growing experimental requirements are still to be addressed. Nevertheless, zfns and talens are capable of correcting gene mutations mediated by hdr repair mechanisms in human ips cells 78 82 and the crisprcas system has also recently been applied to the gene therapy model 50, 83. Cancer genome sequencing has identified numerous somatic mutations whose biologic relevance is uncertain.

Knocking out the activity of a gene provides valuable clues about what that gene normally does. Talenmediated gene disruption on y chromosome reveals. Gene targeting by taleninduced homologous recombination in goats directs production of. However, we failed to detect the obvious atherosclerotic lesions throughout the aortas and their main branches from hypercholesterolemic apoe.

Generation of conditional acvrl1 knockout mice by crisprcas9mediated gene targeting. An alternative to microinjection for creating genetically modified rodents the take electroporation method outperforms microinjection for delivery of. Knockout mice created by talen mediated gene targeting young hoon sung, injeoung baek, duk hyoung kim, jisun jeon, jaehoon lee, kyunghee lee, daewon jeong, jinsoo kim, and hanwoong lee. The gene targeting ability of crisprcas9 in mice was first confirmed by targeting the transgene in enhanced green fluorescent protein egfp transgenic mice. Transcription activatorlike tal effector nucleases talens are repeat domain. The targeted pgcs were germline competent and were used to produce ddx4 null offspring. Mice lacking apoe gene have been the most widely used animal model for atherosclerosis 19 since the first apoe ko mice were produced by classical gene knockout strategy using homologous recombination in es cell with targeting vector 20, 21. Apancreatic pigs cloned using pdx1disrupted fibroblasts.

Bovine or swine, in vitroproduced zygotes were injected with talen mrna on day 1 d1 and cultured. Using the knockout mice method to find relevant gene. In this regard, enormous amount of endeavors was used for generating knockout mice, since embryonic stem cells es cells for homologous recombination or nuclear transfer technologies are established firstly in mice species. Efficient generation of largescale genomemodified mice. Knockout mice created by talenmediated gene targeting to the editor. The crisprcas system has been adapted as an efficient gene targeting technology with the potential for multiplexed genome editing. The first recorded knockout mouse was created by mario r. Onestep generation of mice carrying reporter and conditional. Talen vectors were designed to target exons 1, 3, and 5 of the mouse grk5 gene. Nakamura k, fujii w, tsuboi m, tanihata j, teramoto. Talen mediated gene targeting in avian pgcs is therefore an efficient process. We used gene editing vectors to efficiently disrupt the.

Pdf on jan 9, 20, young hoon sung and others published knockout mice created by talenmediated gene targeting find, read and cite all the research you need on researchgate. Mice carrying mutations in multiple genes are traditionally generated by sequential recombination in embryonic stem cells andor timeconsuming intercrossing of mice with a single mutation. Generation of a monkey with mecp2 mutations by talenbased. Efficient talen mediated myostatin gene editing in goats bmc developmental biology, jul 2016 baoli yu, rui lu, yuguo yuan, ting zhang, shaozheng song, zhengqiang qi, bin shao, mengmin zhu, fei mi, yong cheng. Here, we report the generation of a cynomolgus monkey macaca fascicularis carrying mecp2 mutations using transcription activatorlike effector nucleases talens mediated gene targeting.

Knockout mice created by talenmediated gene targeting. Transcription activatorlike effector nucleases talens are representative nextgeneration platforms for customized genomic editing in transgenic animals, as well as cultured cells in vitro. Here, we created reporter and conditional mutant mice by coinjection of zygotes with cas9 mrna and different guide rnas sgrnas as well as dna vectors of different sizes. Here, we generated specific gene knockout chickens through the use of transcription activatorlike effector nuclease talen mediated gene targeting. Author links open overlay panel minghui li deshou wang. One example is the p53 knockout mouse, which is named after a gene known as p53. Electroporation of ascpf1rnp at the zygote stage is an efficient genome editing method to produce ko mice.