scholarly journals Generation of Fibroblasts Lacking the Sal-like 1 Gene by Using Transcription Activator-like Effector Nuclease-mediated Homologous Recombination

2016 ◽  
Vol 29 (4) ◽  
pp. 564-570
Author(s):  
Se Eun Kim ◽  
Ji Woo Kim ◽  
Yeong Ji Kim ◽  
Deug-Nam Kwon ◽  
Jin-Hoi Kim ◽  
...  
Keyword(s):  
Homologous Recombination ◽  
Transcription Activator

scholarly journals Efficient genome engineering by targeted homologous recombination in mouse embryos using transcription activator-like effector nucleases

2014 ◽  
Vol 5 (1) ◽  
Author(s):  
Daniel Sommer ◽  
Annika E. Peters ◽  
Tristan Wirtz ◽  
Maren Mai ◽  
Justus Ackermann ◽  
...  
Keyword(s):  
Homologous Recombination ◽  
Genome Engineering ◽  
Mouse Embryos ◽  
Transcription Activator

scholarly journals Principles of Genetic Engineering

Genes ◽  
2020 ◽  
Vol 11 (3) ◽  
pp. 291 ◽  
Author(s):  
Thomas M. Lanigan ◽  
Huira C. Kopera ◽  
Thomas L. Saunders
Keyword(s):  
Homologous Recombination ◽  
Genetic Engineering ◽  
Dna Sequences ◽  
Cultured Cells ◽  
Embryonic Stem ◽  
Zinc Finger Nucleases ◽  
Chromosome Break ◽  
Transcription Activator ◽  
Random Integration ◽  
Dna Insertion

Genetic engineering is the use of molecular biology technology to modify DNA sequence(s) in genomes, using a variety of approaches. For example, homologous recombination can be used to target specific sequences in mouse embryonic stem (ES) cell genomes or other cultured cells, but it is cumbersome, poorly efficient, and relies on drug positive/negative selection in cell culture for success. Other routinely applied methods include random integration of DNA after direct transfection (microinjection), transposon-mediated DNA insertion, or DNA insertion mediated by viral vectors for the production of transgenic mice and rats. Random integration of DNA occurs more frequently than homologous recombination, but has numerous drawbacks, despite its efficiency. The most elegant and effective method is technology based on guided endonucleases, because these can target specific DNA sequences. Since the advent of clustered regularly interspaced short palindromic repeats or CRISPR/Cas9 technology, endonuclease-mediated gene targeting has become the most widely applied method to engineer genomes, supplanting the use of zinc finger nucleases, transcription activator-like effector nucleases, and meganucleases. Future improvements in CRISPR/Cas9 gene editing may be achieved by increasing the efficiency of homology-directed repair. Here, we describe principles of genetic engineering and detail: (1) how common elements of current technologies include the need for a chromosome break to occur, (2) the use of specific and sensitive genotyping assays to detect altered genomes, and (3) delivery modalities that impact characterization of gene modifications. In summary, while some principles of genetic engineering remain steadfast, others change as technologies are ever-evolving and continue to revolutionize research in many fields.

scholarly journals Histone deacetylases inhibitor and RAD51 recombinase increase transcription activator-like effector nucleases-mediated homologous recombination on the bovine β-casein gene locus

2020 ◽  
Vol 33 (6) ◽  
pp. 1023-1033
Author(s):  
Da Som Park ◽  
Se Eun Kim ◽  
Deog-Bon Koo ◽  
Man-Jong Kang
Keyword(s):  
T Cells ◽  
Homologous Recombination ◽  
Dna Sequencing ◽  
Histone Deacetylases ◽  
Gene Locus ◽  
Trichostatin A ◽  
Hdac Inhibitors ◽  
T Antigen ◽  
Transcription Activator ◽  
Recombination Efficiency

Objective: The efficiency of the knock-in process is very important to successful gene editing in domestic animals. Recently, it was reported that transient loosening of the nucleosomal folding of transcriptionally inactive chromatin might have the potential to enhance homologous recombination efficiency. The objective of this study was to determine whether histone deacetylases (HDAC) inhibitor and RAD51 recombinase (RAD51) expression were associated with increased knock-in efficiency on the β-casein (<i>bCSN2</i>) gene locus in mammary alveolar-large T antigen (MAC-T) cells using the transcription activator-like effector nucleases (TALEN) system.Methods: MAC-T cells were treated with HDAC inhibitors, valproic acid, trichostatin A, or sodium butyrate for 24 h, then transfected with a knock-in vector, RAD51 expression vector and TALEN to target the <i>bCSN2</i> gene. After 3 days of transfection, the knock-in efficiency was confirmed by polymerase chain reaction and DNA sequencing of the target site.Results: The level of HDAC 2 protein in MAC-T cells was decreased by treatment with HDAC inhibitors. The knock-in efficiency in MAC-T cells treated with HDAC inhibitors was higher than in cells not treated with inhibitors. However, the length of the homologous arm of the knock-in vector made no difference in the knock-in efficiency. Furthermore, DNA sequencing confirmed that the precision of the knock-in was more efficient in MAC-T cells treated with sodium butyrate.Conclusion: These results indicate that chromatin modification by HDAC inhibition and RAD51 expression enhanced the homologous recombination efficiency on the <i>bCSN2</i> gene locus in MAC-T cells.

scholarly journals Autoexcision of Bacterial Artificial Chromosome Facilitated by Terminal Repeat-Mediated Homologous Recombination: a Novel Approach for Generating Traceless Genetic Mutants of Herpesviruses

2010 ◽  
Vol 84 (6) ◽  
pp. 2871-2880 ◽  
Author(s):  
Fuchun Zhou ◽  
Qiuhua Li ◽  
Scott W. Wong ◽  
Shou-Jiang Gao
Keyword(s):  
Homologous Recombination ◽  
Genetic Manipulation ◽  
Artificial Chromosome ◽  
Loxp Site ◽  
Terminal Repeat ◽  
Transcription Activator ◽  
Phenotypic Analysis ◽  
Bac Clone ◽  
Artificial Chromosomes

ABSTRACT Infectious bacterial artificial chromosomes (BACs) of herpesviruses are powerful tools for genetic manipulation. However, the presence of BAC vector sequence in the viral genomes often causes genetic and phenotypic alterations. While the excision of the BAC vector cassette can be achieved by homologous recombination between extra duplicate viral sequences or loxP site-mediated recombination, these methods either are inefficient or leave a loxP site mark in the viral genome. Here we describe the use of viral intrinsic repeat sequences, which are commonly present in herpesviral genomes, to excise the BAC vector cassette. Using a newly developed in vitro transposon-based cloning approach, we obtained an infectious BAC of rhesus rhadinovirus (RRV) strain RRV26-95 with the BAC vector cassette inserted in the terminal repeat (TR) region. We showed that the BAC vector cassette was rapidly excised upon reconstitution in cells predominantly through TR-mediated homologous recombination. Genetic and phenotypic analysis showed that the BAC-excised virus was reversed to wild-type RRV. Using this autoexcisable BAC clone, we successfully generated an RRV mutant with a deletion of Orf50, which encodes a replication and transcription activator (RTA) protein. Together, these results illustrate the usefulness of TR for genetic manipulation of herpesviruses when combined with the novel transposon-based cloning approach.

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