• Journal of Life Science
• /
• v.24 no.12
• /
• pp.1269-1275
• /
• 2014

TALEN is a newly developed gene engineering method to knock out specific genes. It contains a DNA binding domain and a Fok1 nuclease domain in the TALEN plasmid. Therefore, the engineered TALEN construct can bind to any region of genomic DNA and cut the target nucleotide, thereby inducing mutation. In this study, we constructed two TALEN constructs targeted to a protein initiation codon (DBEX2) or the 25th upstream region (DBPR25) to enable mRNA synthesis of SETDB1 HMTase. We performed the TALEN cloning in two steps. The first step was from module vectors to pFUS array vectors. We confirmed successful cloning with a colony PCR experiment and Esp31 restriction enzyme digestion, which resulted in a smear band and a 1 Kb insert band, respectively The second step of the cloning was from a pFUS array vector to a mammalian TALEN expression vector. The engineered TALEN construct was sequenced with specific primers in an expression vector. As expected, a specific array from the module vectors was shown in the sequencing analysis. The specific module sequences were regularly arrayed in every 100 bp, and SETDB1 expression totally disappeared in the TALEN-DBEX2 transfection. PCR amplification targeting of DBEX2 was performed, and the PCR product was digested with a T7E1 restriction enzyme. The expression of SETDB1 was down-regulated in the TALEN-DBPR25 transfection. Morphological changes were also observed in the two TALEN constructs with transfected HeLa cells. These results suggest that the engineered TALEN constructs in two strategic approaches are very useful to knock-out of the SETDB1 gene and to study gene function.

• Asian-Australasian Journal of Animal Sciences
• /
• v.33 no.2
• /
• pp.360-372
• /
• 2020

Objective: Specific genomic sites can be recognized and permanently modified by genome editing. The discovery of endonucleases has advanced genome editing in pigs, attenuating xenograft rejection and cross-species disease transmission. However, off-target mutagenesis caused by these nucleases is a major barrier to putative clinical applications. Furthermore, off-target mutagenesis by genome editing has not yet been addressed in pigs. Methods: Here, we generated genetically inheritable α-1,3-galactosyltransferase (GGTA1) knockout Yucatan miniature pigs by combining transcription activator-like effector nuclease (TALEN) and nuclear transfer. For precise estimation of genomic mutations induced by TALEN in GGTA1 knockout pigs, we obtained the whole-genome sequence of the donor cells for use as an internal control genome. Results: In-depth whole-genome sequencing analysis demonstrated that TALEN-mediated GGTA1 knockout pigs had a comparable mutation rate to homologous recombination-treated pigs and wild-type strain controls. RNA sequencing analysis associated with genomic mutations revealed that TALEN-induced off-target mutations had no discernable effect on RNA transcript abundance. Conclusion: Therefore, TALEN appears to be a precise and safe tool for generating genomeedited pigs, and the TALEN-mediated GGTA1 knockout Yucatan miniature pigs produced in this study can serve as a safe and effective organ and tissue resource for clinical applications.

• Molecules and Cells
• /
• v.39 no.9
• /
• pp.687-691
• /
• 2016

Transcription activator-like effector nucleases (TALENs) are powerful tools for targeted genome editing in diverse cell types and organisms. However, the highly identical TALE repeat sequences make it challenging to assemble TALEs using conventional cloning approaches, and multiple repeats in one plasmid are easily catalyzed for homologous recombination in bacteria. Although the methods for TALE assembly are constantly improving, these methods are not convenient because of laborious assembly steps or large module libraries, limiting their broad utility. To overcome the barrier of multiple assembly steps, we report a one-step system for the convenient and flexible assembly of a 180 TALE module library. This study is the first demonstration to ligate 9 mono-/dimer modules and one circular TALEN backbone vector in a one step process, generating 9.5 to 18.5 repeat sequences with an overall assembly rate higher than 50%. This system makes TALEN assembly much simpler than the conventional cloning of two DNA fragments because this strategy combines digestion and ligation into one step using circular vectors and different modules to avoid gel extraction. Therefore, this system provides a convenient tool for the application of TALEN-mediated genome editing in scientific studies and clinical trials.

• Asian-Australasian Journal of Animal Sciences
• /
• v.29 no.4
• /
• pp.564-570
• /
• 2016

The Sal-like 1 gene (Sall1) is essential for kidney development, and mutations in this gene result in abnormalities in the kidneys. Mice lacking Sall1 show agenesis or severe dysgenesis of the kidneys. In a recent study, blastocyst complementation was used to develop mice and pigs with exogenic organs. In the present study, transcription activator-like effector nuclease (TALEN)-mediated homologous recombination was used to produce Sall1-knockout porcine fibroblasts for developing knockout pigs. The vector targeting the Sall1 locus included a 5.5-kb 5' arm, 1.8-kb 3' arm, and a neomycin resistance gene as a positive selection marker. The knockout vector and TALEN were introduced into porcine fibroblasts by electroporation. Antibiotic selection was performed over 11 days by using $300{\mu}g/mL$ G418. DNA of cells from G418-resistant colonies was amplified using polymerase chain reaction (PCR) to confirm the presence of fragments corresponding to the 3' and 5' arms of Sall1. Further, mono- and bi-allelic knockout cells were isolated and analyzed using PCR-restriction fragment length polymorphism. The results of our study indicated that TALEN-mediated homologous recombination induced bi-allelic knockout of the endogenous gene.

• BMB Reports
• /
• v.49 no.4
• /
• pp.226-231
• /
• 2016

Epstein Barr virus (EBV)-encoded nuclear antigen-1 (EBNA1) plays a pivotal in an EBV episome replication and persistence. Despite considerable attempts, there are no EBV drugs or vaccines. We attempted to eradicate EBV episomes by targeting EBNA1 using the transcription activator-like effector nucleases (TALEN) (E1TN). E1TN-mediated transient knockout (KO) of EBNA1 reduced EBNA1 expression, and caused significant loss of EBV genomes and progressive death of EBV-infected cells. Furthermore, when a mixture of EBV-infected Burkitt's lymphoma (BL) cells and EBV-negative BL cells was targeted by E1TN, EBV-negative cells were counter-selected while most EBV-infected cells died, further substantiating that EBNA1 KO caused selective death of EBV-infected cells. TALEN-mediated transient targeting of EBNA1 attenuated the growth of EBV-infected cells, implicating a possible therapeutic application of E1TN for EBV-associated disorders.

• Asian-Australasian Journal of Animal Sciences
• /
• v.33 no.6
• /
• pp.1023-1033
• /
• 2020

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 (bCSN2) 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 bCSN2 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 bCSN2 gene locus in MAC-T cells.

• Molecules and Cells
• /
• v.39 no.7
• /
• pp.530-535
• /
• 2016

Large conductance calcium-activated potassium (BK) channels participate in many important physiological functions in excitable tissues such as neurons, cardiac and smooth muscles, whereas the knowledge of BK channels in bone tissues and osteoblasts remains elusive. To investigate the role of BK channels in osteoblasts, we used transcription activator-like effector nuclease (TALEN) to establish a BK knockout cell line on rat ROS17/2.8 osteoblast, and detected the proliferation and mineralization of the BK-knockout cells. Our study found that the BKknockout cells significantly decreased the ability of proliferation and mineralization as osteoblasts, compared to the wild type cells. The overall expression of osteoblast differentiation marker genes in the BK-knockout cells was significantly lower than that in wild type osteoblast cells. The BK-knockout osteoblast cell line in our study displays a phenotype decrease in osteoblast function which can mimic the pathological state of osteoblast and thus provide a working cell line as a tool for study of osteoblast function and bone related diseases.

• BMB Reports
• /
• v.45 no.12
• /
• pp.686-692
• /
• 2012

Phenotypic analysis of gene-specific knockout (KO) mice has revolutionized our understanding of in vivo gene functions. As the use of mouse embryonic stem (ES) cells is inevitable for conventional gene targeting, the generation of knockout mice remains a very time-consuming and expensive process. To accelerate the large-scale production and phenotype analyses of KO mice, international efforts have organized global consortia such as the International Knockout Mouse Consortium (IKMC) and International Mouse Phenotype Consortium (IMPC), and they are persistently expanding the KO mouse catalogue that is publicly available for the researches studying specific genes of interests in vivo. However, new technologies, adopting zinc-finger nucleases (ZFNs) or Transcription Activator-Like Effector (TALE) Nucleases (TALENs) to edit the mouse genome, are now emerging as valuable and effective shortcuts alternative for the conventional gene targeting using ES cells. Here, we introduce the recent achievement of IKMC, and evaluate the significance of ZFN/TALEN technology in mouse genetics.

• Molecules and Cells
• /
• v.38 no.6
• /
• pp.475-481
• /
• 2015

Programmable nucleases, which include zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and RNA-guided engineered nucleases (RGENs) repurposed from the type II clustered, regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) system are now widely used for genome editing in higher eukaryotic cells and whole organisms, revolutionising almost every discipline in biological research, medicine, and biotechnology. All of these nucleases, however, induce off-target mutations at sites homologous in sequence with on-target sites, limiting their utility in many applications including gene or cell therapy. In this review, we compare methods for detecting nuclease off-target mutations. We also review methods for profiling genome-wide off-target effects and discuss how to reduce or avoid off-target mutations.

• Journal of Animal Reproduction and Biotechnology
• /
• v.34 no.2
• /
• pp.65-69
• /
• 2019

Since the development of the first genetically-modified mouse, transgenic animals have been utilized for a wide range of industrial applications as well as basic research. To date, these transgenic animals have been used in functional genomics studies, disease models, and therapeutic protein production. Recent advances in genome modification techniques such zinc finger nuclease (ZFN), transcription activator-like effector nucleases (TALEN), and clustered regularly interspaced short palindromic repeats (CRIPSR)-Cas9, have led to rapid advancement in the generation of genome-tailored livestock, as well as experimental animals; however, the development of genome-edited poultry has shown considerably slower progress compared to that seen in mammals. Here, we will focus primarily on the technical strategies for production of transgenic and gene-edited chickens, and their potential for future applications.