222 PRODUCTION OF KNOCKOUT RATS BY USING ZINC FINGER NUCLEASES AND TAL EFFECTOR NUCLEASES

The rat has been used as an important animal for understanding human diseases. Genetically engineered rat strains are used as a human disease model in various research fields. Genetically engineered rat strains are now being routinely produced, not only as transgenic animals but also using gene knockout techniques. Recently, zinc finger nucleases (ZFN) and TAL effector nucleases (TALEN) have enabled editing targeted genes without using embryonic stem cells. These techniques have been applied for production of the knockout and knockin animals. We here studied that the effects of gene targeting by ZFN and TALEN introduced into rat embryos for efficient production of knockout rats. We custom-designed ZFN and TALEN plasmids targeted rat interleukin 2 receptor gamma (Il2rg) gene. Each mRNA was transcribed in vitro from these plasmids. Final concentration of mRNA was adjusted at 10 ng μL–1 in sterilized water for microinjection. Messenger RNA was injected into rat pronuclear stage embryos. The embryos were then cultured in vitro to the 2-cell stage, and were transferred into oviducts of pseudopregnant females. The rate of development of offspring of embryos and effects of editing targeted genes were examined. Of 41 two-cell embryos introduced ZFN after embryo transfer, 9 embryos (22%) developed to offspring. Three offspring (33%) had an edited targeted gene locus. In the embryos introduced TALEN, 30% (6 offspring) of embryos developed to offspring after embryo transfer and all offspring had an edited targeted gene locus. This study demonstrated that the ZFN and TALEN mRNA was active after introduction into rat embryos. Knockout rats could be produced by introduction of ZFN and TALEN into rat embryos. ZFN and TALEN will provide a powerful new approach for targeted gene editing not only in rats but also in other animal species.

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Identification of Off-Target Cleavage Sites of Zinc Finger Nucleases and TAL Effector Nucleases Using Predictive Models

Methods in Molecular Biology - Gene Correction ◽

10.1007/978-1-62703-761-7_24 ◽

2014 ◽

pp. 371-383 ◽

Cited By ~ 3

Author(s):

Eli J. Fine ◽

Thomas J. Cradick ◽

Gang Bao

Keyword(s):

Zinc Finger ◽

Predictive Models ◽

Zinc Finger Nucleases ◽

Cleavage Sites ◽

Tal Effector ◽

Tal Effector Nucleases

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Revealing off-target cleavage specificities of zinc-finger nucleases by in vitro selection

Nature Methods ◽

10.1038/nmeth.1670 ◽

2011 ◽

Vol 8 (9) ◽

pp. 765-770 ◽

Cited By ~ 304

Author(s):

Vikram Pattanayak ◽

Cherie L Ramirez ◽

J Keith Joung ◽

David R Liu

Keyword(s):

Zinc Finger ◽

In Vitro Selection ◽

Zinc Finger Nucleases

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333 ESTABLISHMENT OF NOVEL METHOD FOR REPEATED CONSTRUCTION OF ENGINEERED ZINC FINGER NUCLEASE

Reproduction Fertility and Development ◽

10.1071/rdv25n1ab333 ◽

2013 ◽

Vol 25 (1) ◽

pp. 314

Author(s):

W. Fujii ◽

K. Kano ◽

K. Sugiura ◽

K. Naito

Keyword(s):

Zinc Finger ◽

Gene Knockout ◽

Zinc Finger Nucleases ◽

Cell Stage ◽

Endonuclease Domain ◽

Pcr Products ◽

Novel Method ◽

Recognition Helix ◽

Primer Sets

Zinc finger nucleases (ZFN), which are artificial restriction enzymes consisting of an engineered zinc-finger domain (ZF) and an endonuclease domain, can be used for the induction of site-directed mutation and the efficient generation of gene knockout animals. However, the repeated construction of various ZFN sequences is both expensive and time consuming. In this study, we attempted to establish a novel method for inexpensive and rapid ZFN construction. First, we constructed ZFN against mouse Rosa26 and original mouse Gli3 gene loci using short PCR primer sets (>30 bp), which contained 21 bp of the ZF recognition helix for a specific DNA triplet. We prepared 18 sets of such primers and PCR was performed using one of these primer sets and the partial ZF sequence as a template, which was obtained from the first to second DNA recognition helix of mouse Zif268. The PCR products were joined by overlap-PCR and nested PCR, and then inserted into a vector coding the endonuclease domain of FokI nuclease. By these steps, we successfully synthesised intended ZFN vectors containing 4 to 6 fingers. Next, we evaluated the functions of constructed ZFN. The mRNA of constructed ZFN were transcribed in vitro and injected into the cytoplasm of C57BL/6N zygotes. After 24 h of culture, 2-cell stage embryos were subjected to genomic PCR of the target locus, and the PCR products were directly sequenced. When ZFN mRNA for mouse Rosa26 was injected, 3- to 146-bp deletions were detected in 92.8% of injected embryos. This result was almost the same as previously reported for ZFN, indicating that our novel construction method can synthesise functional ZFN, which work as a site-directed nuclease, and that efficiency was comparable with those constructed by conventional PCR methods using long oligonucleotide sets (60 bp).

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Targeted Killing of Glioblastoma Multiforme In Vivo by IL-13 Zetakine Redirected CTLs Made Glucocorticoid Resistant with Zinc Finger Nucleases.

Blood ◽

10.1182/blood.v110.11.2597.2597 ◽

2007 ◽

Vol 110 (11) ◽

pp. 2597-2597

Author(s):

Andreas Reik ◽

Michael C. Holmes ◽

Yuanyue Zhou ◽

Matthew Mendel ◽

Pei-Qi Liu ◽

...

Keyword(s):

Glioblastoma Multiforme ◽

Glucocorticoid Receptor ◽

Zinc Finger ◽

Zinc Finger Nucleases ◽

Dna Breaks ◽

Adoptive Therapy ◽

Glucocorticoid Hormone ◽

Therapy Strategy

Abstract Genetic modification of cytolytic T-lymphocytes (CTL) for enhancing their functional immunobiology is a promising immunotherapeutic approach for the treatment of cancer and infectious disease. CTLs modified to express a chimeric antigen receptor comprising an extracellular IL13 domain and cytoplasmic CD3 domain (IL13-zetakine) can be re-directed both in vitro and in animal models to target glioblastoma multiforme (GBM), which is characterized by high expression of IL13Ralpha2. Patient-derived IL13-zetakine/HyTK expressing CD8+ CTL clones have entered early stage clinical trials. However, their clinical application is frequently limited in this patient population by the pervasive use of dexamethasone, a potent glucocorticoid analogue employed in the management of cerebral edema. Thus iatrogenic dexamethasone-mediated T-cell functional anergy and apoptosis in these patients is a barrier to realizing the full clinical utility of this adoptive therapy strategy. We hypothesized that knocking out the expression of the glucocorticoid receptor would render therapeutic CTLs resistant to the effects of synthetic glucocorticoids, including dexamethasone. We therefore developed engineered zinc finger nucleases (ZFNs) to specifically disrupt the glucocorticoid receptor (GR) locus in the human genome. ZFNs include the cleavage domain of the restriction enzyme FokI linked to an engineered zinc finger DNA-binding domain and can be designed to cleave a predetermined site in the genome. Natural repair of such DNA breaks via the error-prone non-homologous end joining pathway results in the inactivation of the target gene at frequencies which permit the isolation of knock out clones. Employing adenovirally delivered and transiently expressed ZFNs targeting exon 3 of the human GR gene, we isolated IL13-zetakine+ CD8+T-cells containing a biallelically mutated GR locus. These cells were characterized by the absence of full length GR protein, lack of glucocorticoid hormone-induced gene regulation and resistance to glucocorticoid hormone-mediated immunosupression and apoptosis. Importantly, the ZFN-modified, glucocorticoid-resistant CTLs demonstrated zetakine re-directed cytolytic activity and tumor cell specificity in chromium release assays in vitro and in an orthotopic mouse model of GBM in vivo. These results indicate that glucocorticoid-resistant IL13-zetakine targeted CTLs should retain function in cancer patients receiving glucocorticoids. A clinical trial to test this hypothesis is currently under development.

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Genome Editing in Medicine: Tools and Challenges

Acta medica Lituanica ◽

10.15388/amed.2021.28.2.8 ◽

2021 ◽

Vol 28 (2) ◽

pp. 8

Author(s):

Gunda Petraitytė ◽

Eglė Preikšaitienė ◽

Violeta Mikštienė

Keyword(s):

Genome Editing ◽

Viral Vectors ◽

Biological Therapy ◽

Zinc Finger Nucleases ◽

Target Cells ◽

Biological Processes ◽

Tal Effector ◽

Bioethical Issues ◽

Tal Effector Nucleases ◽

Editing Process

Studies which seek fundamental, thorough knowledge of biological processes, and continuous advancement in natural sciences and biotechnology enable the establishment of molecular strategies and tools to treat disorders caused by genetic mutations. Over the years biological therapy evolved from using stem cells and viral vectors to RNA therapy and testing different genome editing tools as promising gene therapy agents. These genome editing technologies (Zinc finger nucleases, TAL effector nucleases), specifically CRISPR-Cas system, revolutionized the field of genetic engineering and is widely applied to create cell and animal models for various hereditary, infectious human diseases and cancer, to analyze and understand the molecular and cellular base of pathogenesis, to find potential drug/treatment targets, to eliminate pathogenic DNA changes in various medical conditions and to create future “precise medication”. Although different concerning factors, such as precise system delivery to the target cells, efficacy and accuracy of editing process, different approaches of making the DNA changes as well as worrying bioethical issues remain, the importance of genome editing technologies in medicine is undeniable. The future of innovative genome editing approach and strategies to treat diseases is complicated but interesting and exciting at once for all related parties – researchers, clinicians, and patients.

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