Site-Directed Homologous Recombination in Tobacco Cell Cultures via Zinc Finger Nucleases

2007 ◽  
pp. 111-113 ◽  
Author(s):  
Charles Q. Cai ◽  
W. Michael Ainley ◽  
Trevor N. Collingwood ◽  
Robbi J. Garrison ◽  
Lisa L. Schulenberg ◽  
...  
Keyword(s):  
Homologous Recombination ◽  
Zinc Finger ◽  
Cell Cultures ◽  
Zinc Finger Nucleases ◽  
Tobacco Cell

scholarly journals High-frequency homologous recombination in plants mediated by zinc-finger nucleases

2005 ◽  
Vol 44 (4) ◽  
pp. 693-705 ◽  
Author(s):  
David A. Wright ◽  
Jeffrey A. Townsend ◽  
Ronnie Joe Winfrey ◽  
Phillip A. Irwin ◽  
Jyothi Rajagopal ◽  
...  
Keyword(s):  
Homologous Recombination ◽  
Zinc Finger ◽  
High Frequency ◽  
Zinc Finger Nucleases

236 GENE TARGETING USING ZINC FINGER NUCLEASES (ZFN) IN THE PORCINE FIBROBLAST

2012 ◽  
Vol 24 (1) ◽  
pp. 230
Author(s):  
S. Kim ◽  
J. W. Kim ◽  
S. M. Lee ◽  
J. H. Kim ◽  
M. J. Kang
Keyword(s):  
Homologous Recombination ◽  
Gene Targeting ◽  
Zinc Finger ◽  
Marker Gene ◽  
Somatic Cells ◽  
Embryonic Stem ◽  
Domestic Animals ◽  
Zinc Finger Nucleases ◽  
Exogenous Dna ◽  
Targeting Vector

Gene targeting is a genetic technique that utilises homologous recombination between an engineered exogenous DNA fragment and the endogenous genome of an animal. In domestic animals, gene targeting has provided an important tool for producing knockout pigs for the α1,3-galactosyltransferase gene (GGTA1) to use in xenotransplantation. The frequency of homologous recombination is a critical parameter for the success of gene targeting. The efficiency of homologous recombination in somatic cells is lower than that in mouse embryonic stem cells. The application of gene targeting to somatic cells has been limited by its low efficiency. Recently, knockout rat and mouse were generated by introducing nonhomologus end joining (NHE)-mediated deletion or insertion at the target site using zinc-finger nucleases (ZFN). Therefore, the development of effective knockout and knock-in techniques in domestic animals is very important in biomedical research. In this study, we investigated homologous recombination events at the cytidine monophospho-N-acetylneuraminic acid hydroxylase (CMAH) gene locus using ZFN in porcine primary fibroblast. The CMAH-targeted ZFN plasmid and mRNA were purchased from Sigma-Aldrich (St Louis, MO, USA). Porcine ear fibroblasts cells were obtained from a 10-day-old male Chicago miniature pigs. The fibroblasts were cultured in DMEM containing 15% fetal bovine serum, 1 × nonessential amino acids, 1 × sodium pyruvate, 10–4 M β-mercaptoethanol, 100 unit mL–1 penicillin and 100 μg mL–1 streptomycin. The cells were trypsinized and resuspended at a concentration of 1.25 × 107 cells mL–1 in F10 nutrient mixture. Four hundred microliters of the cell suspension was electroporated in a 4-mm cuvette with 4 pulses of 1 ms duration using 400V capacitive discharges using the CMAH neo targeting vector and ZFN plasmid or RNA. The CMAH neo targeting vector consists of the neomycin resistance gene (neo) as a positive selectable marker gene, 789-bp 5′ arm and 763-bp 3′ arm from exon 8 of CMAH gene. After selection of G-418, PCR analysis was performed using 64 colonies transfected with ZFN plasmid and 48 colonies transfected with ZFN RNA. As a result, 19 positive colonies were identified in colonies transfected with ZFN plasmid and 15 colonies were identified in colonies transfected with ZFN RNA. The targeting efficiency was 29.7 and 31.6% in the colonies transfected with ZFN plasmid and ZFN RNA, respectively. To our knowledge, this study provides the first evidence that the efficiency of gene targeting using ZFN was higher than that of conventional gene targeting in the porcine fibroblast. These cell lines may be used in production of CMAH knockouts for xenotransplantation.

332 COMBINATION OF ZINC FINGER NUCLEASES AND MODIFIED BACTERIAL ARTIFICIAL CHROMOSOMES REVEALS TREMENDOUS RATES OF HOMOLOGOUS RECOMBINATION

2013 ◽  
Vol 25 (1) ◽  
pp. 314
Author(s):  
P. Fezert ◽  
A. Wuensch ◽  
E. Wolf ◽  
N. Klymiuk
Keyword(s):  
Homologous Recombination ◽  
Zinc Finger ◽  
Reporter Gene ◽  
Strand Break ◽  
Double Strand Break ◽  
Double Strand Breaks ◽  
Zinc Finger Nucleases ◽  
Strand Breaks ◽  
Artificial Chromosomes ◽  
Targeting Vector

DNA-based vectors have been used for decades to modify the genomes of mammalian cells by homologous recombination in a specific and site-directed way. Even though various modifications of the procedure have been presented, efficiency is relatively low for many target sites and novel projects still have an unforeseeable outcome. This is in particularly true for site-directed mutagenesis in primary cells intended for use in the generation of large animal models because of their impaired predisposition for homologous recombination compared with stem cells. The recent development of site-specific nucleases is based on a completely different principle: they do not necessarily involve recombination between DNA strands, but rather make use of the inefficient correction of double-strand breaks in the genomic DNA by the cellular DNA repair machinery after such a double-strand break has been introduced by a synthetic enzyme that directed nuclease activity to a defined site in the genome. Here, we intended to evaluate the potential of zinc finger nucleases (ZFN) to introduce a lacZ reporter gene into the CFTR locus. Initially, the efficiency of 3 different ZFN pairs was examined under different conditions revealing modification efficiencies between 0 and 38%. An optimized protocol was used to combine the most efficient ZFN pair with either a bacterial artificial chromosome (BAC) vector or a conventional targeting vector carrying the desired modification. Although the conventional vector failed to introduce the reporter gene in any of more than 200 clones examined, the BAC correctly modified the target site in 32 of 75 clones in a heterozygous way and in 10 out of 75 clones in a homozygous way. However, the introduction of small vector fragments into the CFTR locus in rare cases indicated that the ZFN caused a double-strand break but the vector was not able to act as a recombination donor. On the other hand, transfection of the BAC alone only resulted in 1 modified clone out of 98 and, thus, our data strongly support the hypothesis that the forced introduction of double-strand breaks dramatically increases the rate of homologous recombination, but they also provide indication that the design of the targeting vector has a profound influence on the efficiency.

scholarly journals Gene targeting by homologous recombination in mouse zygotes mediated by zinc-finger nucleases

2010 ◽  
Vol 107 (34) ◽  
pp. 15022-15026 ◽  
Author(s):  
M. Meyer ◽  
M. H. de Angelis ◽  
W. Wurst ◽  
R. Kuhn
Keyword(s):  
Homologous Recombination ◽  
Gene Targeting ◽  
Zinc Finger ◽  
Zinc Finger Nucleases ◽  
Mouse Zygotes

scholarly journals Gene Correction by Homologous Recombination With Zinc Finger Nucleases in Primary Cells From a Mouse Model of a Generic Recessive Genetic Disease

2010 ◽  
Vol 18 (6) ◽  
pp. 1103-1110 ◽  
Author(s):  
Jon P Connelly ◽  
Jenny C Barker ◽  
Shondra Pruett-Miller ◽  
Matthew H Porteus
Keyword(s):  
Homologous Recombination ◽  
Mouse Model ◽  
Zinc Finger ◽  
Genetic Disease ◽  
Zinc Finger Nucleases ◽  
Primary Cells ◽  
Gene Correction
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