Knockout mice created by talen-mediated gene targeting pdf

However, many of the resulted founder animals were chimeric ones with multiple mutations through the embryo injection 17. Although thousands of genes have been disrupted in mice by conventional gene targeting 6, investigators growing experimental requirements are still to be addressed. 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. Application of oocyte cryopreservation technology in talen. 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. Pdf grk5knockout mice generated by talenmediated gene. Knockout rat models mimicking human atherosclerosis created by cpf1mediated gene targeting. 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. Es cell gene targeting technique to be defective in a particular gene product provide an attractive model system in which to. 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. 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. In this study, we used genomeediting tools to create and analyze targeted somatic mutations in murine models of liver cancer.

Developing genetically engineered mouse models using. 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. Knockout mice created by talen mediated gene targeting to the editor. Transcription activatorlike effector nucleases talens are a new type of engineered nuclease that is very effective for directed gene disruption in any genome sequence. Claudin4 knockout by talenmediated gene targeting in. Electroporation of ascpf1rnp at the zygote stage is an efficient genome editing method to produce ko mice. The type ii bacterial crisprcas system is a novel genomeengineering technology with the ease of multiplexed gene targeting. Efficient biallelic gene knockout and sitespecific knock. But, gene targeting in human pluripotent stem cells including es and ips cells has been very difficult historically.

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. Department of biochemistry, college of life science. 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. Knockout mice created by talen mediated gene targeting.

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. Talenmediated gene disruption on y chromosome reveals. 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. Phenotypic analysis of genespecific knockout mice has transformed our understanding of in vivo gene functions. Transcription activatorlike tal effector nucleases talens are repeat domain. 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. Efficient talenmediated gene knockout in livestock. G proteincoupled receptor kinase 5 grk5 is one of the seven grk family members whose primary function is to desensitize g proteincoupled receptors gpcrs. Genome editing in mice using tale nucleases request pdf. 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. 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. We discuss how conventional gene targeting by homologous.

The first recorded knockout mouse was created by mario r. An alternative to microinjection for creating genetically modified rodents the take electroporation method outperforms microinjection for delivery of. 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. 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. Efficient talenmediated myostatin gene editing in goats. A, left and right target sequences of the mstn gene for d10a. 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. However, we failed to detect the obvious atherosclerotic lesions throughout the aortas and their main branches from hypercholesterolemic apoe.

The primary goal of this study was to establish an atherosclerotic rat model. Insights derived from ace knockout mice justin cole, dilek ertoy, kenneth e bernstein keywords. Sitespecific knockin is another advantage of nucleasemediated gene targeting in large animals. Recent advances in gene editing technology have introduced the potential for application of mutagenesis approaches in nonhuman primates to model human development and disease. Transcription activatorlike effector nucleases talens are representative nextgeneration platforms for customized genomic editing in transgenic animals, as. Knocking out the activity of a gene provides valuable clues about what that gene normally does. Gene targeting by taleninduced homologous recombination in goats directs production of. The gene targeting ability of crisprcas9 in mice was first confirmed by targeting the transgene in enhanced green fluorescent protein egfp transgenic mice. Talenmediated gene mutagenesis in rhesus and cynomolgus. Generation of conditional acvrl1 knockout mice by crispr. Gene engineering for generating targeted mouse mutants is a key technology for biomedical research. Request pdf genome editing in mice using tale nucleases gene engineering for generating targeted mouse mutants is a key technology for biomedical research. The efficiency of all sgrnas was first tested by cotransfection with cas9 into the cos7 cell line derived from african green monkey kidney.

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. Recently, two new genetargeting technologies have been developed. Doublestrand dna breaks dramatically enhance hr 10. Talen mediated endogenous gene disruption using oocytes from female mice at different weeks of age. Knockout mice created by talenmediated gene targeting to the editor. There are several limiting factors of conventional gene targeting in mice, such as the timeconsuming process of targeting vector construction, low spontaneous hr. 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. Efficient generation of largescale genomemodified mice.

Here, we report the generation of a cynomolgus monkey macaca fascicularis carrying mecp2 mutations using transcription activatorlike effector nucleases talens mediated gene targeting. Grk5knockout mice generated by talenmediated gene targeting. The feasibility of talenmediated gene disruption has been confirmed in creating gene knockout mice and rats recently. 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. 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. 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.

Transcription activatorlike effector nucleases talens are representative nextgeneration platforms for customized genomic editing in transgenic animals, as well as cultured cells in vitro. Pdf knockout mice created by talenmediated gene targeting. The value of knockout mouse models a detailed analysis. 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. The apoerat was created by talen mediated gene targeting in the genetic background of sprague dawley rat. Jul 27, 2016 myostatin mstn encodes a negative regulator of skeletal muscle mass that might have applications for promoting muscle growth in livestock.

We used gene editing vectors to efficiently disrupt the. Aspects of the technology for generating knockout mice, and the mice themselves have been patented in many countries by private companies. Anephrogenic phenotype induced by sall1 gene knockout in. 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. These techniques target the detailed study of gene activity.

Knockout rat models mimicking human atherosclerosis created. 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. Generation of knockout mice, however, remains a timeconsuming and expensive process. 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. Production of sry knockout mouse using talen via oocyte. Here, we report the generation of a cynomolgus monkey macaca fascicularis carrying mecp2 mutations using transcription activatorlike effector nucleases talensmediated gene targeting. Talen mediated gene targeting in avian primordial germ cells is therefore an efficient process. Sixto eightweekold male rats were used in the experiments n 10 in each group. 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. Importantly, the crisprinduced tumors had features. 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. Cancer genome sequencing has identified numerous somatic mutations whose biologic relevance is uncertain.

Crisprcas9 technique was able to efficiently modify alk1 and create the target mice. Using the knockout mice method to find relevant gene. Efficient talen mediated gene knockout in livestock. In this study, we accomplished targeted knockout of the ovalbumin ov gene in the chicken. Onestep generation of mice carrying mutations in multiple. 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. Transcription activatorlike effector nucleases talen were designed against. Onestep generation of mice carrying reporter and conditional. Knockout rat models mimicking human atherosclerosis. Dec 27, 20 gene targeting technologies with zinc finger nucleases zfns, transcription activator. Sung yh, baek ij, kim dh, jeon j, lee j, lee k, jeong d, kim js, lee hw. We amputated all four limbs of each animal to examine the effect of tsp.

Targeted gene deletion of mirnas in mice by talen system. All the aforementioned initiatives have the possibility to contribute to remarkable new treatments that could save many lives. Highefficiency and heritable gene targeting in mouse by. Transcriptomics of gabra4 knockout mice reveals common nmdar pathways underlying autism, memory, and epilepsy cuixia fan, yue gao, guanmei liang, lang huang, jing wang. Apolipoprotein edeficient rats develop atherosclerotic. Highly efficient targeted mutagenesis in onecell mouse embryos. 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. Talen mediated gene targeting in avian pgcs is therefore an efficient process. Talenmediated somatic mutagenesis in murine models of cancer. Efficient talenmediated myostatin gene editing in goats pdf. Here we report successful talen mediated mutagenesis of an xlinked, rett. Apancreatic pigs cloned using pdx1disrupted fibroblasts.

Talenmediated gene targeting in avian pgcs is therefore an efficient process. We have created a strain of mice that lacks the y subtype of pkc to evaluate. Applying the method to knock in a series of cry1 mutants into cry1. Cpf1 mediated generation of apoe andor ldlr knockout rats. To target these genes in the mouse genome, we designed and synthesized highly active. Genome editing in mice using tale nucleases springerlink. Generating mouse models for biomedical research disease.

Schematic representation of the genetic engineering methods used for generating targeted knockout rats. Claudin4 knockout by talenmediated gene targeting in mdck cells. Screening methods to identify talenmediated knockout mice. Our results extended the application of stateoftheart talen technology from experimental animals to farm animals. To explore the regionspecific genome functions in vivo, generation of gene targeting animals is a powerful strategy. The most commonly used method consists of three major steps. After injecting talens mrna into monkey zygotes achieved by in vitro. Generation of a monkey with mecp2 mutations by talenbased. The targeted pgcs were germline competent and were used to produce ddx4 null offspring. In many model systems, this approach has been invaluable for discovering the function of ge. Capecchi, martin evans, and oliver smithies in 1989, for which they were awarded the 2007 nobel prize in physiology or medicine. Genetargeted mutant animals, such as knockout or knockin mice, have dramatically improved our. Knockout mice created by talenmediated gene targeting generation of knockout mice, however, remains a timeconsuming and expensive process. Knockout mice created by talenmediated gene targeting.

We investigated the generation of mice with genetic knockout ko of the g proteincoupled receptor kinase grk 5 gene by microinjection of talen mrna. Gene knockout ko mice are powerful tools for studying biological science and genetic diseases of humans skarnes et al. Conventional embryonic stem es cell mediated gene targeting requires at least 6 months to obtain homozygous mutant mice. Author links open overlay panel minghui li deshou wang. Targeted gene knockout by editing specific loci in genome has revolutionized the field of functional genomics. Bovine or swine, in vitroproduced zygotes were injected with talen mrna on day 1 d1 and cultured. Timeline and generation of mutant mice by single plasmid injection. Efficient talenmediated gene knockout in livestock pnas.

Generation of genemodified cynomolgus monkey via cas9rna. Electroporation of ascpf1rnp at the zygote stage is an. Targeted gene knockout in chickens mediated by talens pnas. Phenotypic analysis of gene specific knockout mice has transformed our understanding of in vivo gene functions.

Pdf transcription activatorlike effector nucleases talens are programmable. Gene editing nuclease and its application in tilapia. Oct 23, 2012 transcription activatorlike effector nucleases talens are programmable nucleases that join foki endonuclease with the modular dnabinding domain of tales. 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. The crisprcas system has been adapted as an efficient gene targeting technology with the potential for multiplexed genome editing. Generation of conditional acvrl1 knockout mice by crisprcas9mediated gene targeting. 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.

This lecture about transgenic animals explains the mechanism of gene knockout mice production. We investigated the generation of mice with genetic knockout ko of the g proteincoupled receptor kinase grk 5 gene by microinjection of. In this study, we report the detailed phenotype of eif2s3y knockout mice generated using talenmediated gene disruption. Recently, a taleninduced y chromosomal gene modification in embryonic stem cells and oocytes was successfully utilized to generate knockout mice 14,15. Talen vectors were designed to target exons 1, 3, and 5 of the mouse grk5 gene. In ddx4 knockout chickens, pgcs are initially formed but are lost during meiosis in the developing ovary, leading to adult female sterility. To our knowledge, this is first report of targeted mutagenesis in monkeys with talen technology for modeling of human disease. Gene targeting by taleninduced homologous recombination. Here, we generated specific gene knockout chickens through the use of transcription activatorlike effector nuclease talen mediated gene targeting. Gene editing in model organisms has provided critical insights into brain development and diseases. Pdf efficient talenmediated gene knockout in livestock. T1 knockout mice created by talen mediated gene targeting.

Transcriptomics of gabra4 knockout mice reveals common nmdar pathways underlying autism, memory, and epilepsy. As talenmediated knockout chickens are genetically modified but nontransgenic, without genomic integration. The convenience of this technique means that t alen mediated. 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. Nakamura k, fujii w, tsuboi m, tanihata j, teramoto. Targeted gene knockout in chickens mediated by talens. Grk5knockout mice generated by talenmediated gene targeting article pdf available in animal biotechnology 274. Crisprcas9based genome editing in mice by single plasmid. One example is the p53 knockout mouse, which is named after a gene known as p53. In ddx4 knockout chickens, pgcs are initially formed but are lost during meiosis in the developing ovary leading to adult female sterility.