Design, construction and in vitro testing of zinc finger nucleases

2006 ◽  
Vol 1 (3) ◽  
pp. 1329-1341 ◽  
Author(s):  
Dana Carroll ◽  
J Jason Morton ◽  
Kelly J Beumer ◽  
David J Segal
Keyword(s):  
Zinc Finger ◽  
Zinc Finger Nucleases ◽  
In Vitro Testing ◽  
Design Construction

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

2011 ◽  
Vol 8 (9) ◽  
pp. 765-770 ◽  
Author(s):  
Vikram Pattanayak ◽  
Cherie L Ramirez ◽  
J Keith Joung ◽  
David R Liu
Keyword(s):  
Zinc Finger ◽  
In Vitro Selection ◽  
Zinc Finger Nucleases

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

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).

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

Blood ◽  
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.

scholarly journals Design, construction, and analysis of specific zinc finger nucleases for microphthalmia - associate transcription factor

2012 ◽  
Vol 55 (4) ◽  
pp. 559-568 ◽  
Author(s):  
Wenwen Wang ◽  
Hongmao Liu ◽  
Haibin Zhao ◽  
Zaili Luo ◽  
Yongxiang Shi
Keyword(s):  
Transcription Factor ◽  
Zinc Finger ◽  
Zinc Finger Nucleases ◽  
Design Construction

scholarly journals Engineering of macaque CD4+ T cells and CD34+ hematopoietic stem cells resistant to in vitro SIV infection using zinc finger nucleases

2016 ◽  
Vol 2 ◽  
pp. 15-16
Author(s):  
Peter Lentz ◽  
Gregory Bonello ◽  
Fuchun Zhou ◽  
Mary Salas ◽  
Magdalena Cepeda ◽  
...  
Keyword(s):  
Stem Cells ◽  
T Cells ◽  
Hematopoietic Stem Cells ◽  
Zinc Finger ◽  
Cd4 T Cells ◽  
Zinc Finger Nucleases ◽  
Hematopoietic Stem ◽  
Siv Infection

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

2014 ◽  
Vol 26 (1) ◽  
pp. 225
Author(s):  
T. Kaneko ◽  
T. Mashimo
Keyword(s):  
Embryo Transfer ◽  
Zinc Finger ◽  
Gene Locus ◽  
Genetically Engineered ◽  
Zinc Finger Nucleases ◽  
Rat Strains ◽  
Rat Embryos ◽  
Tal Effector ◽  
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.

Myostatin knockout using zinc-finger nucleases promotes proliferation of ovine primary satellite cells in vitro

2014 ◽  
Vol 192 ◽  
pp. 268-280 ◽  
Author(s):  
Fatemeh Salabi ◽  
Mahmood Nazari ◽  
Qing Chen ◽  
Jonathan Nimal ◽  
Jianming Tong ◽  
...  
Keyword(s):  
Zinc Finger ◽  
Satellite Cells ◽  
Zinc Finger Nucleases

Urokinase Evaluated with the Experimental Radioactive Embolus

1967 ◽  
Vol 17 (03/04) ◽  
pp. 405-411
Author(s):  
M Hume
Keyword(s):  
Animal Experiment ◽  
Blood Clot ◽  
In Vitro Testing ◽  
Effective Agent

SummaryUrokinase and urokinase-activated plasmin have been given to the dog and rabbit. A thrombolytic state has been induced. Purified urokinase has induced lysis of the experimental radioactive blood clot embolus in the circulation. Demonstration of effectiveness in this animal experiment is hampered by inhibition of the agents in the circulation to a degree much greater than was noted in previous experiments with streptokinase. In vitro testing indicates that under proper conditions urokinase will be an effective agent in the treatment of human thromboembolism.

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