Generation of a triple-gene knockout mammalian cell line using engineered zinc-finger nucleases

2010 ◽  
pp. n/a-n/a ◽  
Author(s):  
Pei-Qi Liu ◽  
Edmond M. Chan ◽  
Gregory J. Cost ◽  
Lin Zhang ◽  
Jianbin Wang ◽  
...  
Keyword(s):  
Cell Line ◽  
Zinc Finger ◽  
Mammalian Cell ◽  
Gene Knockout ◽  
Zinc Finger Nucleases ◽  
Mammalian Cell Line

329 GENETIC TARGETING OF THE PORCINE α1,3-GALACTOSYLTRANSFERASE GENE IN FETAL FIBROBLAST CELLS USING ZINC FINGER NUCLEASES

2011 ◽  
Vol 23 (1) ◽  
pp. 260 ◽  
Author(s):  
J. Hauschild ◽  
A. L. Queisser ◽  
J. W. Carnwath ◽  
G. Cost ◽  
Y. Santiago ◽  
...  
Keyword(s):  
Cell Line ◽  
Zinc Finger ◽  
Cell Population ◽  
Magnetic Beads ◽  
Catalytic Domain ◽  
Gene Knockout ◽  
Fluorescein Isothiocyanate ◽  
Zinc Finger Nucleases ◽  
Fetal Fibroblast ◽  
Donor Cells

Hyperacute rejection after porcine-to-human xenotransplantation is caused by binding of preformed human antibodies against Gal-epitopes on the surface of porcine cells. Organs from Gal-negative pigs have shown prolonged survival after transplantation into baboons. Knocking out a gene by conventional gene targeting frequency is extremely inefficient (homologous recombination = 0.0001 to 0.001%; Denning et al. 2001). Recent publications in rats (Geurts et al. 2009) show that the gene knockout via zinc finger nuclease (ZFN)-driven nonhomologous end joining (NHEJ) can be enhanced 10 000-fold over conventional approaches, making it feasible to generate a biallelic gene knockout with one ZFN application. Here, we used ZFN technology to generate porcine cells that carry a ZFN-mediated knockout of the Gal gene to use these cells as donor cells in somatic cell nuclear transfer (SCNT) to obtain live offspring. One primary porcine fetal fibroblast cell line was transfected by electroporation (n = 6) with a pair of ZFN plasmids designed to target the DNA sequence encoding the catalytic domain located in exon 9 of the α1,3-gal locus. Transfected cells were incubated (7 days at a combination of 30°C and 37°C) and analysed for Gal expression by fluorescence activated cell sorting (FACS) using fluorescein isothiocyanate (FITC)-conjugated isolectin-B4. On average, 1.4% (± 0.3%; n = 6) of the cells were free of Gal epitopes, indicating a biallelic knockout. DNA mutation detection analysis (Cel-I assay) of cell cultures gave a mean frequency of 3.5% NHEJ (± 1.3%; n = 6) giving the fraction of mutant alleles within the cell population. One cell line with 1% Gal-negative cells was sorted by a magnetic Dynabead-based separation method to select for Gal-negative cells (Fujimura et al. 2008). Because of the limited amount of Gal-negative cells within the cell population, we chose to select the cells with magnetic beads. This method is gentler to the cells and leads to a higher plating efficiency after sorting compared with FACS. The sorted cells could be easily expanded and will serve as donor cells in SCNT to show the feasibility of generating knockout pigs via ZFN-mediated gene knockout. This study demonstrates that ZFN technology is an applicable tool to produce genetically modified porcine cells for use as donors in SCNT and to speed the creation of pig models for xenotransplantation and human diseases.

Production of myostatin gene knockout Wuzhishan miniature pig fibroblasts with zinc-finger nucleases

2013 ◽  
Vol 35 (6) ◽  
pp. 778-785
Author(s):  
Sui-Zhong CAO ◽  
Cheng-He YUE ◽  
Xi-Rui LI ◽  
Chong FENG ◽  
Chuan LONG ◽  
...  
Keyword(s):  
Zinc Finger ◽  
Gene Knockout ◽  
Zinc Finger Nucleases ◽  
Miniature Pig ◽  
Myostatin Gene

Pharmacology of human dopamine D3 receptor expressed in a mammalian cell line: comparison with D2 receptor

1992 ◽  
Vol 225 (4) ◽  
pp. 331-337 ◽  
Author(s):  
Pierre Sokoloff ◽  
Marc Andrieux ◽  
Roger Besançon ◽  
Catherine Pilon ◽  
Marie-Pascale Martres ◽  
...  
Keyword(s):  
Cell Line ◽  
Mammalian Cell ◽  
D2 Receptor ◽  
Dopamine D3 Receptor ◽  
D3 Receptor ◽  
Mammalian Cell Line ◽  
Dopamine D3

Instability of simple sequence repeats in a mammalian cell line

1994 ◽  
Vol 3 (2) ◽  
pp. 253-256 ◽  
Author(s):  
Rosann A. Farber ◽  
Thomas D. Petes ◽  
Margaret Dominska ◽  
Sarah S. Hudgens ◽  
R.Michael Liskay
Keyword(s):  
Cell Line ◽  
Mammalian Cell ◽  
Simple Sequence Repeats ◽  
Mammalian Cell Line ◽  
Simple Sequence

Radiosensitization of a Mammalian Cell Line with 5-Bromodeoxyuridine

1962 ◽  
Vol 17 (4) ◽  
pp. 479 ◽  
Author(s):  
N. Delihas ◽  
M. A. Rich ◽  
M. L. Eidinoff
Keyword(s):  
Cell Line ◽  
Mammalian Cell ◽  
Mammalian Cell Line

scholarly journals Construction and Modelling of an Inducible Positive Feedback Loop Stably Integrated in a Mammalian Cell-Line

2011 ◽  
Vol 7 (6) ◽  
pp. e1002074 ◽  
Author(s):  
Velia Siciliano ◽  
Filippo Menolascina ◽  
Lucia Marucci ◽  
Chiara Fracassi ◽  
Immacolata Garzilli ◽  
...  
Keyword(s):  
Cell Line ◽  
Mammalian Cell ◽  
Positive Feedback ◽  
Feedback Loop ◽  
Mammalian Cell Line ◽  
Positive Feedback Loop

scholarly journals Production of hypoallergenic milk from DNA-free beta-lactoglobulin (BLG) gene knockout cow using zinc-finger nucleases mRNA

2018 ◽  
Vol 8 (1) ◽  
Author(s):  
Zhaolin Sun ◽  
Ming Wang ◽  
Shiwen Han ◽  
Shuangyu Ma ◽  
Zhiyuan Zou ◽  
...  
Keyword(s):  
Zinc Finger ◽  
Gene Knockout ◽  
Zinc Finger Nucleases ◽  
Beta Lactoglobulin

The use of ‘Omics technology to rationally improve industrial mammalian cell line performance

2015 ◽  
Vol 113 (1) ◽  
pp. 26-38 ◽  
Author(s):  
Amanda M. Lewis ◽  
Nicholas R. Abu-Absi ◽  
Michael C. Borys ◽  
Zheng Jian Li
Keyword(s):  
Cell Line ◽  
Mammalian Cell ◽  
Mammalian Cell Line ◽  
Omics Technology

scholarly journals Expression of Recombinant Non Structural 1 Protein of Dengue Virus Serotype-2 in Mammalian Cell Line

2019 ◽  
Vol 13 (1) ◽  
pp. 36-42 ◽  
Author(s):  
FITHRIYAH SJATHA ◽  
◽  
OKTIVIA CHANDRA MUSTIKA ◽  
ANGKY BUDIANTI ◽  
TJAHJANI MIRAWATI SUDIRO ◽  
...  
Keyword(s):  
Dengue Virus ◽  
Cell Line ◽  
Mammalian Cell ◽  
Mammalian Cell Line ◽  
Virus Serotype ◽  
Dengue Virus Serotype 2
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