Polyethylenimine-Mediated CCR5 Gene Knockout Using Transcription Activator-Like Effector Nucleases

2018 ◽  
Vol 14 (3) ◽  
pp. 546-552 ◽  
Author(s):  
Lian Jin ◽  
Yan Deng ◽  
Nongyue He ◽  
Lijun Wang ◽  
Mengling Weng
Keyword(s):  
Gene Knockout ◽  
Transcription Activator ◽  
Ccr5 Gene

scholarly journals Gene regulation of adult skeletogenesis in starfish and modifications during gene network co-option

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Atsuko Yamazaki ◽  
Shumpei Yamakawa ◽  
Yoshiaki Morino ◽  
Yasunori Sasakura ◽  
Hiroshi Wada
Keyword(s):  
Sea Urchins ◽  
Gene Knockout ◽  
Expression Patterns ◽  
Homeobox Gene ◽  
Regulatory System ◽  
Nutrient Sensing ◽  
Regulatory Genes ◽  
Transcription Activator ◽  
Patiria Pectinifera ◽  
Skeleton Formation

AbstractThe larval skeleton of the echinoderm is believed to have been acquired through co-option of a pre-existing gene regulatory network (GRN); that is, the mechanism for adult skeleton formation in the echinoderm was deployed in early embryogenesis during echinoderm diversification. To explore the evolutionary changes that occurred during co-option, we examined the mechanism for adult skeletogenesis using the starfish Patiria pectinifera. Expression patterns of skeletogenesis-related genes (vegf, vegfr, ets1/2, erg, alx1, ca1, and clect) suggest that adult skeletogenic cells develop from the posterior coelom after the start of feeding. Treatment with inhibitors and gene knockout using transcription activator-like effector nucleases (TALENs) suggest that the feeding-nutrient sensing pathway activates Vegf signaling via target of rapamycin (TOR) activity, leading to the activation of skeletogenic regulatory genes in starfish. In the larval skeletogenesis of sea urchins, the homeobox gene pmar1 activates skeletogenic regulatory genes, but in starfish, localized expression of the pmar1-related genes phbA and phbB was not detected during the adult skeleton formation stage. Based on these data, we provide a model for the adult skeletogenic GRN in the echinoderm and propose that the upstream regulatory system changed from the feeding-TOR-Vegf pathway to a homeobox gene-system during co-option of the skeletogenic GRN.

Genome Editing Toolbox

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. SCI-11-SCI-11
Author(s):  
Andrew M. Scharenberg
Keyword(s):  
Genome Engineering ◽  
Gene Knockout ◽  
Zinc Finger Nucleases ◽  
Practical Implementation ◽  
Homing Endonucleases ◽  
Gene Repair ◽  
Dna Breaks ◽  
Transcription Activator ◽  
Advisory Committees ◽  
Core Technology

Abstract Nucleases capable of making targeted breaks in genomic DNA are a core technology required for genome engineering, an emerging field of technology for making precise alterations in cellular genomes. Over the past ten years, four major platforms have emerged for generation of nucleases able to make targeted DNA breaks with a high degree of efficiency and specificity: homing endonucleases, zinc finger nucleases, transcription activator-like (TAL) effector nucleases, and RNA-guided nucleases. This talk will cover the biochemistry and platform-specific attributes of each type of nuclease, along with evolution/improvements in nucleases and related technologies and aspects of the practical implementation of nuclease technology for gene knockout and gene repair in primary hematopoietic cells. Disclosures Scharenberg: Pregenen Inc.: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Cellectis therapeutics: Consultancy.

scholarly journals A Highly Efficient and GMP Compliant Protocol to Manufacture CCR5 Edited Cells to Treat HIV Infection

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 5795-5795
Author(s):  
Marianna Romito ◽  
Emily Meyer ◽  
Sushmita Poddar ◽  
Helena Heinz ◽  
Julia Rositzka ◽  
...  
Keyword(s):  
T Cells ◽  
Cd4 T Cells ◽  
Research Funding ◽  
Transcription Activator ◽  
Ccr5 Gene ◽  
Hematopoietic Stem ◽  
Highly Efficient ◽  
Proliferation Capacity

Abstract Targeted genome editing in blood and immune cells enable new therapeutic applications, especially for infectious diseases. We present a GMP-compliant protocol to manufacture CCR5-edited CD34+ hematopoietic stem and precursor cells (HSPCs) with the goal to cure patients suffering from chronic infection with human immunodeficiency virus type 1 (HIV1). We hypothesize that genetic disruption of the CCR5 gene, which encodes the major HIV1 co-receptor, in HSPCs will give rise to an HIV-resistant immune system after transplantation. We have developed engineered nucleases based on transcription activator-like effector nucleases (TALENs) targeting CCR5. Electroporation of CD4+ T-cells and CD34+ HSPCs with mRNAs encoding TALENs revealed disruption of up to 80% of CCR5 alleles in CD4+ T-cells and over 90% of alleles in HSPCs. The high gene editing frequencies in T-cells and HSPCs were confirmed by deep sequencing, and no cleavage activity above background levels were detected at the top 20 predicted off-target sites. CCR5-edited CD4+ cells preserved their proliferation capacity and their biological function. Importantly, these cells showed significantly reduced CCR5 expression and became resistant to infection with the R5-tropic HIV-1JR-FL virus. The CCR5-edited HSPCs maintained their proliferation potential and their capacity to differentiate into the various blood lineages in vitro and in vivo, and clonal analysis revealed bi-allelic CCR5 disruption in more than 75% of cells. In summary, our developed protocol enables highly efficient and GMP-compliant knockout of the CCR5 locus in clinically relevant cells, so forming the foundation for a planned phase I/II clinical study. Disclosures Gautron: Cellectis SA: Employment. Busser:Cellectis: Employment, Patents & Royalties: Cellectis. Smith:Cellectis. Inc: Employment, Patents & Royalties. Duchateau:Cellectis: Employment, Patents & Royalties: Cellectis. Cathomen:TRACR Hematology: Consultancy; Cellectis: Research Funding; Miltenyi Biotec: Research Funding. Cornu:Cellectis: Research Funding; Miltenyi Biotec: Research Funding.

337 NONMEIOTIC INTROGRESSION OF QUANTITATIVE TRAIT NUCLEOTIDES AND CORRECTION OF CONGENITAL MUTATIONS IN LIVESTOCK WITH TRANSCRIPTION ACTIVATOR-LIKE EFFECTOR NUCLEASES

2013 ◽  
Vol 25 (1) ◽  
pp. 316
Author(s):  
S. C. Fahrenkrug ◽  
W. Tan ◽  
S. G. Lillico ◽  
D. Stverakova ◽  
C. Proudfoot ◽  
...  
Keyword(s):  
Quantitative Trait ◽  
Gene Editing ◽  
Production Systems ◽  
Gene Knockout ◽  
Direct Injection ◽  
Genetic Enhancement ◽  
Transcription Activator ◽  
Tal Effectors ◽  
Cattle Breeds ◽  
Specific Allele

Genetic enhancement of livestock productivity and welfare are major goals of breeding and genetics programs. However, the introgression of desirable alleles across breeds is slow and inaccurate. The development of gene editing technologies would provide the opportunity to accelerate the genetic improvement of a diversity of livestock breeds. Transcription activator-like effector nucleases (TALEN) are programmable nucleases that join the modular DNA binding domain of transcription activator-like (TAL) effectors with FokI endonuclease. We found that TALEN could be easily manufactured and that 64% displayed activity in swine and cattle primary fibroblasts, with cleavage of 1.5 to 45% of chromosomes in cell populations, as measured by Surveyor assay. Clonal isolation and sequencing revealed that up to 84% of cells contained at least one modified allele, with up to 24% of cells containing biallelic or homozygous chromosomal modification. Co-transfection of a customized TALEN pair with a template containing a specific allele was effective at the nonmeiotic introgression of quantitative trait into naïve cattle breeds. We will also describe the repair of 2 recently described embryonic lethal mutations that are segregating in important dairy cattle breeds (JH1 and HH1). Injection of TALEN mRNA into the cytoplasm of pig and cattle zygotes was capable of inducing gene knockout (KO) in 27 to 75% of embryos analysed (n = 4–59), nearly half of which (8/19) harbored biallelic modification. We will present data describing efforts towards gene conversion by direct injection of livestock embryos. Finally, we will present alternative strategies for the incorporation of gene editing in livestock production systems by cloning or embryo treatment.

31 PRODUCTION EFFICIENCY OF GENE KNOCKOUT PIGS USING GENOME EDITING AND SOMATIC CELL CLONING

2015 ◽  
Vol 27 (1) ◽  
pp. 108
Author(s):  
H. Matsunari ◽  
M. Watanabe ◽  
K. Nakano ◽  
A. Uchikura ◽  
Y. Asano ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Genome Editing ◽  
Production Efficiency ◽  
Gene Knockout ◽  
Genetically Modified ◽  
Zinc Finger Nucleases ◽  
International Institute ◽  
Induced Mutations ◽  
Transcription Activator

Genome editing technologies have been used as a powerful strategy for the generation of genetically modified pigs. We previously developed genetically modified clone pigs with organogenesis-disabled phenotypes, as well as pigs exhibiting diseases with similar features to those of humans. Here, we report the production efficiency of various gene knockout cloned pigs from somatic cells that were genetically modified using zinc finger nucleases (ZFN) or transcription activator-like effector nucleases (TALEN). The ZFN- or TALEN-encoding mRNAs, which targeted 7 autosomal or X-linked genes, were introduced into porcine fetal fibroblast cells using electroporation. Clonal cell populations carrying induced mutations were selected after limiting dilution. The targeted portion of the genes was amplified using PCR, followed by sequencing and mutation analysis. Among the collected knockout cell colonies, cells showing good proliferation and morphology were selected and used for somatic cell nuclear transfer (SCNT). In vitro-matured oocytes were obtained from porcine cumulus-oocyte complexes cultured in NCSU23-based medium and were used to obtain recipient oocytes for SCNT after enucleation. SCNT was performed as reported previously (Matsunari et al. 2008). The cloned embryos were cultured for 7 days in porcine zygote medium (PZM)-5 to assess their developmental ability. Cloned embryos were transplanted into the oviduct or uterus of oestrus-synchronized recipient gilts to evaluate their competence to develop to fetuses or piglets. Cloned embryos reconstructed with 7 types of knockout cells showed equal development to blastocysts compared with those derived from the wild-type cells (54.5–83.3% v. 60.7%). Our data (Table 1) demonstrated that the reconstructed embryos derived from knockout cells could efficiently give rise to cloned offspring regardless of the type of genome editing methodology (i.e. ZFN or TALEN). Table 1.Production efficiency of gene knockout cloned pigs using genome editing This study was supported by JST, ERATO, the Nakauchi Stem Cell and Organ Regeneration Project, JST, CREST, Meiji University International Institute for Bio-Resource Research (MUIIBR), and JSPS KAKENHI Grant Number 26870630.

scholarly journals Use of the heteroduplex mobility assay and cell sorting to select genome sequences of the CCR5 gene in HEK 293T cells edited by transcription activator-like effector nucleases

2014 ◽  
Vol 37 (1) ◽  
pp. 120-126 ◽  
Author(s):  
Arildo Nerys-Junior ◽  
Lendel C. Costa ◽  
Luciene P. Braga-Dias ◽  
Márcia Oliveira ◽  
Átila D. Rossi ◽  
...  
Keyword(s):  
Cell Sorting ◽  
Transcription Activator ◽  
Genome Sequences ◽  
Ccr5 Gene ◽  
Heteroduplex Mobility Assay

Abstract 229: Searching Genetic Modifiers For bag3 -based Cardiomyopathy Using Adult Zebrafish Models

2017 ◽  
Vol 121 (suppl_1) ◽  
Author(s):  
Yonghe Ding ◽  
Alexey Dvornikov ◽  
Xiao Ma ◽  
Hong Zhang ◽  
Xiaolei Xu
Keyword(s):  
Cardiac Dysfunction ◽  
Gene Knockout ◽  
Individualized Medicine ◽  
Genetic Modifier ◽  
Fish Mortality ◽  
Genetic Modifiers ◽  
Adult Zebrafish ◽  
Transcription Activator ◽  
Inherited Cardiomyopathy ◽  
Forward Mutagenesis

We recently developed a forward mutagenesis screening strategy in adult zebrafish to screen gene-breaking transposon (GBT) mutants, and identified four genetic modifiers for doxorubicin-induced cardiomyopathy. However, it remains unclear whether these genetic modifiers identified from an acquired cardiomyopathy model exert similar modifying effects on inherited cardiomyopathy models. To address this question, we generated BCL2-associated athanogene 3 (bag3) gene knockout in adult zebrafish, using the transcription activator-like effector nucleases (TALEN) genome editing technology. In the bag3-/- fish, progressive cardiac phenotypes reminiscent of human cardiomyopathy such as fetal gene activation, myofibril loss and cardiac dysfunction were detected. At the single myofibril level, reduced active contractility was observed, supporting the dilated cardiomyopathy (DCM)-like phenotype. Based on the ejection fraction index quantified using a newly developed ex vivo assay, different pathogenesis stages including pre-DCM, early-DCM and late DCM were defined. Next, we assessed the potential modifying effects of the four DIC-modifying mutants on bag3 -based cardiomyopathy model. Different from the other three GBT mutants, GBT0411+/- , which tags the long isoform of dnajb6 b gene, dramatically accelerated the cardiac dysfunction and fish mortality in the bag3-/- fish, suggesting dnajb6b as a sensitive genetic modifier for bag3-based cardiomyopathy. Mechanistically, we showed that Bag3 physically interacts with Dnajb6, and we hypothesize that impaired autophagy and/or endoplasmic reticulum stress convey the synergistic cardiac dysfunction and fish mortality phenotypes in the GBT0411+/-;bag3-/- double mutants. In summary, this study demonstrates that an inherited cardiomyopathy model can be established in an adult zebrafish, which can be utilized to search genetic modifiers. Future studies employing this simple vertebrate model amenable to forward mutagenesis screening promise systematic identification of genetic modifiers for different types of cardiomyopathies, a foundation for individualized medicine.

scholarly journals The kiss/kissr Systems Are Dispensable for Zebrafish Reproduction: Evidence From Gene Knockout Studies

Endocrinology ◽  
2014 ◽  
Vol 156 (2) ◽  
pp. 589-599 ◽  
Author(s):  
Haipei Tang ◽  
Yun Liu ◽  
Daji Luo ◽  
Satoshi Ogawa ◽  
Yike Yin ◽  
...  
Keyword(s):  
Gene Knockout ◽  
Functional Redundancy ◽  
Signaling System ◽  
Transcription Activator ◽  
Neuroendocrine Control ◽  
Paralogous Genes ◽  
Reproductive Capability ◽  
Mutant Lines

The kiss1/gpr54 signaling system is considered to be a critical regulator of reproduction in most vertebrates. However, this presumption has not been tested vigorously in nonmammalian vertebrates. Distinct from mammals, multiple kiss1/gpr54 paralogous genes (kiss/kissr) have been identified in nonmammalian vertebrates, raising the possibility of functional redundancy among these genes. In this study, we have systematically generated the zebrafish kiss1−/−, kiss2−/−, and kiss1−/−;kiss2−/− mutant lines as well as the kissr1−/−, kissr2−/−, and kissr1−/−;kissr2−/− mutant lines using transcription activator-like effector nucleases. We have demonstrated that spermatogenesis and folliculogenesis as well as reproductive capability are not impaired in all of these 6 mutant lines. Collectively, our results indicate that kiss/kissr signaling is not absolutely required for zebrafish reproduction, suggesting that the kiss/kissr systems play nonessential roles for reproduction in certain nonmammalian vertebrates. These findings also demonstrated that fish and mammals have evolved different strategies for neuroendocrine control of reproduction.

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