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Ait Boujmaa, Mohamed, und Houssam Khelalfa. „Geotechnical Stability Analysis of the Quay Wall of Military Port Ksar Sghir, Morocco“. Journal of Earth and Marine Technology (JEMT) 2, Nr. 2 (30.03.2022): 73–78. http://dx.doi.org/10.31284/j.jemt.2022.v2i2.2374.
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This article consists in establishing a geotechnical stability study of the -4m/zh quay belonging to the Ksar Sghir Port. First of all, using the TALREN software for verification against large slips. the study presents a verification of the stability for the rear slope slip circles and for the front slope slip circles in the static case. Secondly, stability was determined for the back slope slip circles and for the front slope slip circles in the seismic case. Moreover, PLAXIS software was used to model the displacements and constraints in the structure. Following the results of the safety coefficients, we notice that the stability of the structure gives acceptable values. In addition, the soil does not reach breaking stress. The total mean stress and total displacement mostly impacted the surface area that is related to external impact.
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Inabi, Omar, Mustapha Attou, Mostafa Benzaazoua und Mohamed Qachar. „Design of Cost-Effective and Sustainable Treatments of Old Landslides Adapted to the Moroccan Road Network: A Case Study of Regional Road R410 Crossing the Rifan Structural Domain“. Water 15, Nr. 13 (30.06.2023): 2423. http://dx.doi.org/10.3390/w15132423.
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The Moroccan road network is susceptible to multiple landslides annually, particularly in the northern regions due to high rainfall and specific geology. These events result in significant economic and social negative consequences, highlighting the need for sustainable and cost-effective solutions for network maintenance. This study outlines the methodology employed in addressing the issues within the RR410 regional road (Rifain region of Morocco), which entailed a thorough examination of the malfunctions, specific surveys, laboratory testing, and problem modeling. By incorporating long-term test-derived shear strength parameters, the model indicated that the road platform was stable, and back analysis using TALREN 4 software allows for model calibration. At kilometric point 23, using earthwork-based solutions (e.g., purging and replacing the base layer, employing granular water-insensitive substitution material) was found to provide a sustainable alternative to the expensive reinforced concrete-based solutions commonly used. Furthermore, these solutions contributed to the use of environmentally friendly and locally sourced materials. Road alignment rectification to anchor the platform in suitable soil was also an effective solution, as demonstrated at kilometric point 48. Additionally, enhancing the drainage and sanitation infrastructure, such as installing draining trenches, spurs, and reinforcing existing water structures, is a crucial aspect of addressing most landslides in the region.
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Pandey, Dipak, Takahiro Matsubara, Taiju Saito, Yukinori Kazeto, Koichiro Gen, Tetsushi Sakuma, Takashi Yamamoto, Miyuki Mekuchi und Rie Goto. „TALEN-Mediated Gene Editing of slc24a5 (Solute Carrier Family 24, Member 5) in Kawakawa, Euthynnus affinis“. Journal of Marine Science and Engineering 9, Nr. 12 (04.12.2021): 1378. http://dx.doi.org/10.3390/jmse9121378.
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Transcription activator-like effector (TALE) nucleases (TALENs) mediated gene editing methods are becoming popular and have revealed the staggering complexity of genome control during development. Here, we present a simple and efficient gene knockout using TALENs in kawakawa, Euthynnus affinis, using slc24a5. We examined slc24a5 gene expression and functional differences between two TALENs that hold the TALE scaffolds, +153/+47 and +136/+63 and target slc24a5. Developmental changes in slc24a5 transcripts were seen in early-stage embryos by real-time PCR; slc24a5 expression was first detected 48 h post fertilization (hpf), which increased dramatically at 72 hpf. Four TALENs, 47- and 63-type of two different target loci (A and B), respectively, were constructed using Platinum TALEN and evaluated in vitro by a single-strand annealing (SSA) assay. TALEN activities were further evaluated in vivo by injecting TALEN mRNAs in the two-cell stage of the zygote. Most of the TALEN-induced mutants showed mosaic patterns in the retinal pigment epithelium (RPE) and fewer melanin pigments on the body at 72 hpf and later when compared to the control, implying the gene’s association with melanin pigment formation. A heteroduplex mobility assay (HMA) and the genome sequence further confirmed the TALEN-induced mutations of substitution, insertion, and deletion at an endogenous locus.
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Heigwer, Florian, Grainne Kerr, Nike Walther, Kathrin Glaeser, Oliver Pelz, Marco Breinig und Michael Boutros. „E-TALEN: a web tool to design TALENs for genome engineering“. Nucleic Acids Research 41, Nr. 20 (03.09.2013): e190-e190. http://dx.doi.org/10.1093/nar/gkt789.
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Lee, Sanghoon, Changhong Yin, Janet Ayello, Carmella van de Ven, Henry Hwang, Charles Biggar, James Pao, Erin Mulvey und Mitchell S. Cairo. „Talens-Mediated DLEU1 Gene Silencing in Burkitt Lymphoma (BL): Implication of Lncrna DLEU1 Gene As a Potential Tumor Suppressor Gene in BL.“ Blood 120, Nr. 21 (16.11.2012): 2403. http://dx.doi.org/10.1182/blood.v120.21.2403.2403.
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Abstract Abstract 2403 Background: Pediatric Burkitt Lymphoma (BL) is the most common histological subtype of Non Hodgkin Lymphoma (NHL) in children and adolescents (Cairo et al., Blood, 2007; Miles/Cairo, BJHaem., 2012). We previously identified in a subset analysis that children with BL and a 13q deletion, particularly 13q14.3, had significantly poorer outcome and inferior overall survival (OS) despite aggressive short, intensive multiagent chemotherapy (Poirel/Cairo et al., Leukemia, 2009; Nelson/Cairo/Sanger et al., BJHaem., 2009). Deleted in Lymphocytic Leukemia 1 (DLEU1) is a long non codign RNA gene with the BL classifier genes as reported by Dave et al (NEJM. 2006) on chromosome 13q14.3 region and the expression levels of RASSF1, TUBB2C and UBR1 were significantly higher in BL than in diffuse large B-cell lymphoma (DLBCL) (Day/Cairo et al., AACR, 2008). Sequence-specific Zinc Finger Nucleases (ZFN) and Transcription Activator-Like Effector Nucleases (TALENs) technologies have been developed for precision targeted genome editing in in vitro experiments with high efficiency and TALEN technology has been applied in a variety of organisms and stem cells for new experimental and therapeutic tools (Carlson et al., Mol Ther., 2012). Objectives: We hypothesize that 1) TALEN technology is suitable for the modification of and silencing of DLEU1 locus and 2) DLEU1 may have function as a tumor suppressor gene and therefore examined whether the loss of DLEU1 by TALEN-mediated DLEU1 knockdown Raji BL cells result in change in proliferation and programmed cell death. Methods: TALENs were constructed based on REAL (restriction enzyme and ligation) assembly methods (Sander et al., Nat. Biotech, 2011) for DLEU1 gene modification. DLEU1 TALEN pairs or combinations were transfected into 293T and Raji cells using lipofectamin reagent (Invitrogen) and Amaxa nucleofection kit, respectively. Genomic DNA and total RNA were extracted using genomic DNA extraction kit (Promega) and Trizol reagent (Invitrogen). Surveyor mutation detection assay (Transgenomic) was applied for TALENs validities and confirmed by sequencing analysis. Quantitative RT-PCR was performed by CFX96 Real-time system (Bio-rad) using qScript™ cDNA Synthesis Kit (Quantas) and SsoFast™ EvaGreen® Supermix (Bio-rad). Transfected cells were re-plated (1×104) into 48 well plates at 24hours post transfection and then counted every 24hrs for cell proliferation assay. For spontaneous apoptosis assay, DLEU1 TALENs transfected Raji cells were re-plated into 96 well plates at 24hours post-transfection and measured caspase 3/7 activities (Promega) using Clarity Luminescence microplate reader (Biotek) and statistical significance was determined by a one or two-tailed Student t-test. Results: Three pairs of functional DLEU1 TALENs (T1, T2, and T3) targeting endogenous DLEU1 gene generated and confirmed their validities with gene modification caused by non-homologous end joining (NHEJ) in TALENs mediated 293T [Figure 1]. Forty eight hours after transiently transfection in Raji cells, combined DLEU1 TALENs, T1/T2 (T12), T1/T3 (T13) and T2/T3 (T23) mediated Raji cells were dramatically reduced expression of DLEU1 mRNA (about 82–93% reduction) compared to untransfected Raji cells [92% reduction (p<0.002), 93% (p<0.0006), and 92% (p<0.0003)], respectively, and compared to empty vector as a control [82% reduction (p<0.05), 87% (p<0.0001), and 82% (p<0.001, respectively)]. T12 and T13-mediated DLEU1 knockdown Raji cells showed significantly an increase of cell proliferation [1.2-fold (p<0.02) and 1.5-fold (p<0.04) at day 5, respectively]. In spontaneous apoptotic assays, T13 mediated Raji showed significantly 20% (p<0.05) of reduction of caspase 3/7 activities. In comparison of mRNA expression of DLEU1 network genes in T13 mediated Raji cells, there were no significant differences in mRNA expression of c-myc compared to control, however, there was significantly reduced expression of RASSF1 and UBR1 mRNA (<7.7-fold, p<0.005 and <11.1-fold, p<0.0005), respectively, and significantly increased expression of TUBB2C mRNA (>7.5-fold, p<0.005). Conclusions: We demonstrate that 1) DLEU1-TALENs are useful tools for the modification of endogenous DLEU1 gene expression, 2) DLEU1 may have as a tumor suppressor gene in BL, and 3) DLEU1 TALEN mediated gene knockdown resulted in inhibition of BL apoptosis and increase in cell proliferation. Disclosures: No relevant conflicts of interest to declare.
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Wang, Song, Yan Yu, Yuhualei Pan, Huan Wang, Yushang Zhao, Mengyao Qu und Yanbing Zhu. „Simple and efficient custom transcription activator-like effector gene synthesis via twin primer assembly“. BioTechniques 70, Nr. 2 (Februar 2021): 100–106. http://dx.doi.org/10.2144/btn-2020-0130.
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Transcription activator-like effector (TALE) nucleases (TALENs) efficiently recognize and cleave DNA in a sequence-dependent manner. However, current TALE custom synthesis methods are either complicated or expensive. Here we report a simple and low-cost method for TALE construct assembly. This method utilizes the denaturation/reannealing nature of double-stranded DNA to create a unique single-stranded DNA overhang for proper ordering of TALE monomers in an engineered multimer. We successfully synthesized two TALEN pairs targeting the endogenous TET1 locus in human embryonic kidney cells and demonstrated their editing efficiency. Our method provides an alternative simple, low-cost method for effective TALEN assembly, which may improve the application of TALE-based technology.
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Virgilio, Maria, Grzegorz Pietka und Elspeth M. Payne. „Ribosomal Proteins Rps19 and Rps14 Cooperate As Tumor Suppressor Genes with p53“. Blood 124, Nr. 21 (06.12.2014): 2943. http://dx.doi.org/10.1182/blood.v124.21.2943.2943.
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Abstract Diamond-Blackfan Anemia (DBA) is a congenital bone marrow failure syndrome that manifests as a profound macrocytic anemia and classically presents within the first year of life. Heterozygous mutations in, or genomic loss of one of several Ribosomal Protein (RP) genes have been identified in over 50% of DBA patients, most commonly RPS19, accounting for 25% of all cases. DBA shares a similar erythroid phenotype to the 5q- subtype of myelodysplastic syndrome in which anemia is thought to arise from heterozygous loss of RPS14. Anemia in these conditions is at least partially due to p53-mediated apoptosis and cell cycle arrest of erythroid progenitors. To further study the role of p53 in the pathogenesis of DBA and 5q- syndrome, we employed genome editing tools to generate stable Rps14 and Rps19 knockout zebrafish lines. We generated Transcription Activator-Like Effector Nucleases (TALENs) targeting exon 1 of rps19 and exon 1 of rps14 as well as Clustered, Regularly Interspaced, Short Palindromic Repeats (CRISPR) single guide RNAs (sgRNA) targeting exon 2 of rps19. TALENs or CRISPRs were injected into p53m214k/m214k zebrafish embryos at the single-cell stage. This zebrafish line carries a mutated p53 that is insensitive to DNA damage and hence prone to tumor formation. rps19 CRISPR sgRNAs were injected with mRNAs encoding Cas9, Cas9D10A nickase, and a ssDNA guide with a human DBA mutation. For each cohort of embryos injected, genomic DNA analysis from 20 phenotypically normal embryos from each clutch was screened to determine the efficacy of cleavage by TALEN and CRISPR using MiSeq. Mutations were identified in 30% (rps14 TALEN) 29% (rps19 TALEN), 27% (rps19 Crispr Cas9) and 12% (rps19 Crispr Cas9D10A) of reads. None of the rps19Crispr Cas9D10A carried the ssDNA guide mutation, rather single nucleotide variants and indels similar to those observed with Cas9. The remaining embryos from each F0 clutch were raised in order to generate stable mutant lines in the F1 generation; however, early, overt tumor growth was noted in all RP injected lines. Tumors were observed from 4 months post fertilization compared with 9 months for uninjected controls. F0 RP mosaic fish continued to develop tumors earlier than uninjected counterparts. At 10 months of age tumor development was statistically significantly higher in rps19 and rps14 TALEN and rps19 Cas9D10A and trended towards significance in rps19 Cas9 injected fish. Overall survival was significantly reduced in each of the cohorts compared to p53m214k/m214k uninjected controls (p<0.0001). Preliminary histology of grown tumors has shown melanomas and malignant peripheral nerve sheath tumors. Zebrafish injected with an unrelated TALEN targeting a zinc transporter (SLC30A10), into p53m214k mutant embryos do not show any increase in tumor formation compared to uninjected controls. Notably several RP’s have been shown to be haploinsufficient tumor suppressor genes in their own right in zebrafish and drosophila models. To determine if the early tumor development in p53m214k zebrafish was simply additive to a potential tumor suppressor effect of Rps14 or Rps19 alone, we injected WT embryos with the rps14 and rps19 TALENS. High mortality in rps14 and rps19 TALEN injected WT embryos impeded this analysis; however recent published reports on stable Rps19 mutant zebrafish do not report an increase in tumor incidence. Interestingly, embryo survival was not affected when TALENs were injected into p53m214/+. Analysis of these zebrafish is ongoing. Our results show that loss of Rps14 or Rps19 accelerates the development of tumors in the p53m214k/m214k mutant line. This effect is independent of the RP or the method of mutation (TALEN vs CRISPR), indicating that off target effects are unlikely to be responsible for this observation. As these are mosaic F0 fish, it is possible that tumors may arise from cells with homozygous RP mutations. Further molecular analysis will reveal this. We have now identified 2 stable Rps19 mutant lines, and tumor analysis of F1 fish from these lines is ongoing. In conclusion, we have shown that loss of Rps14 or Rps19 cooperates with a loss of function p53 mutation to accelerate tumor formation and death. Our results highlight the importance of caution in using p53 suppressors as a therapeutic option in RP deficient patients. Disclosures No relevant conflicts of interest to declare.
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Navas, Patrick A., Yongqi Yan, Minerva E. Sanchez, Ericka M. Johnson und George Stamatoyannopoulos. „Talen-Mediated Knock Outs Of Cis and Trans Elements Potentially Involved In Globin Gene Switching“. Blood 122, Nr. 21 (15.11.2013): 436. http://dx.doi.org/10.1182/blood.v122.21.436.436.
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Abstract Transcription activator-like effector nucleases (TALEN) are engineered proteins used for precise genome editing by generating specific DNA double strand that are repaired by homologous recombination and by non-homologous end joining. TALENs can be used to study gene regulation by deleting putative regulatory elements in the context of the native chromosome and measuring mRNA synthesis. We designed TALENs to delete individual DNAse I-hypersensitive sites (HS) of the β-globin locus control region (LCR) followed by an assessment of globin gene expression and assessment of epigenetic effects in K562 erythroleukemia cells. The β-globin LCR is composed of five HSs and functions as a powerful regulatory element responsible for appropriate levels of the five β-like globin genes during development. Introduction of plasmid DNA encoding a pair of TALENs and targeting individually the flanking region of the HS2, HS3 and HS4 core elements along with a donor 100 base single-stranded oligonucleotide resulted in the successful deletions of each of the three core elements in K562 cells. Individual K562 cells were seeded to produce clones and the mutations were screened by PCR to identify both heterozygous and homozygous clones. The TALEN-mediated 288 bp HS2 core deletion resulted 32 heterozygous (48.5%) and 6 homozygous clones (9.1%) in a total of 66 clones screened. K562 carries three copies of chromosome 11 emphasizing the robustness of TALEN technology to target each of the alleles. In the 199 bp HS3 core deletion, from 113 clones we identified 28 heterozygous (24.8%) and 3 (2.7%) homozygous clones. Lastly, the 301 bp HS4 core deletion yielded 9 homozygous (5.9%) and 12 heterozygous (7.9%) clones from 151 clones screened. Total RNA was isolated from wild-type K562 cells, and from both the heterozygous and homozygous mutant clones and subjected to RNase Protection analysis to quantitate the levels of globin mRNA. Deletion if the HS3 core in K562 cells in a ∼30% reduction in ε-globin mRNA and 2-fold reduction in γ-globin mRNA. A more dramatic effect on globin expression is observed in the HS2 core deletion, as ε- and γ-globin expression is reduced by 2- and 5-fold, respectively. These results suggest that HS2 contributes the majority of the LCR enhancer function in K562 cells. The HS4 core deletion resulted in a modest ∼20% reduction in both ε- and γ-globin expression. TALENs were designed to knockout trans-acting factors implicated to be involved in globin gene regulation and/or globin switching. TALENs bracketing the gene promoters and the first exon of 25 genes encoding either a transcription factor or histone-modifying enzyme were synthesized and post-transfection PCR screens of the transfected pool of K562 cells resulted in the successful identification of 17 gene knockouts. The 17 target genes are PRMT5, LDB1, EIF2AK3, BCL11A, HBSIL, MYB, SOX6, NFE4, NR2F2, NR2C1, NR2C2, CHTOP, NFE2, DNMT3A, RBBP4, MTA2 and MBD2. Single cell clones have been generated by limited dilution of transfected K562 pools and thus far we have identified heterozygous and homozygous clones of 8 of 17 gene knockouts, importantly all clones were identified without selection. The frequency of identifying the knockout clones, represented by the number of clones screened/ number of heterozygous clones/ number of homozygous clones, are as follows: HBS1L (63/3/0), SOX6 (68/13/2), NFE4 (56/13/7), LBD1 (300/2/0), MBD2 (301/0/1), CHTOP (288/66/6), NFE2 (712/44/5) and NR2C1 (96/40/11). The remaining nine gene knockouts and globin gene expression data will be presented at the meetings. These studies highlight a powerful TALEN-mutagenesis platform for target deletions of both cis- and trans-elements to study globin gene switching. TALENs can be synthesized in several days and the screening of the individual clones for the desired knockouts is completed within two weeks. This highly efficient mutagenesis platform will further our understanding of the molecular basis of globin switching. Disclosures: No relevant conflicts of interest to declare.
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Chen, Shiu-Jau, und Yuan-Chuan Chen. „Potential Application of TALENs against Murine Cytomegalovirus Latent Infections“. Viruses 11, Nr. 5 (03.05.2019): 414. http://dx.doi.org/10.3390/v11050414.
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Cytomegalovirus (CMV) infections are still a global health problem, because the latent viruses persist in humans and cause recurring diseases. Currently, there are no therapies for CMV latent infections and the therapies for active infections are limited by side effects and other problems. It is impossible to eradicate latent viruses in animals. HCMV (human CMV) is specific to human diseases; however, it is difficult to study HCMV due to its host specificity and long life cycle. Fortunately, MCMV (murine CMV) provides an excellent animal model. Here, three specific pairs of transcription activator-like effector nuclease (TALEN) plasmids (MCMV1–2, 3–4, and 5–6) were constructed to target the MCMV M80/80.5 sequence in order to test their efficacy in blocking MCMV lytic replication in NIH3T3 cell culture. The preliminary data showed that TALEN plasmids demonstrate specific targeting and cleavage in the MCMV M80/80.5 sequence and effectively inhibit MCMV growth in cell culture when the plasmid transfection is prior to the viral infection. The most specific pairs of TALEN plasmids (MCMV3–4) were further used to confirm the negative regulation of latent MCMV replication and gene expression in Balb/c mice. The injection of specific TALEN plasmids caused significant inhibition in the copy number level of immediately early gene (ie-1) DNA in five organs of mice, when compared with the controls. The result demonstrated that TALENs potentially provide an effective strategy to remove latent MCMV in animals.
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Khudoyorova, Munira. „TALMEN ART IN NAVOI CONTINENTS“. International Journal Of Literature And Languages 03, Nr. 05 (01.05.2023): 12–15. http://dx.doi.org/10.37547/ijll/volume03issue05-03.
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The article discusses the art of talmeh used in the continents included in Alisher Navoi's book "Khazayin ul-Maoni". Special attention is paid to the role of Talmeh in illuminating the essence of the continents. Continents with characters such as Qorun, Hamza, Yusuf, Jesus, and Mary were analysed.
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Zenner, Eline, und Reinhild Vandekerckhove. „Talen geven en talen nemen“. Taal en Tongval 69, Nr. 1 (01.09.2017): 1–15. http://dx.doi.org/10.5117/tet2017.1.zenn.
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Fahrenkrug, S. C., S. G. Lillico, C. Proudfoot, T. J. King, J. H. Pryor, C. R. Long, C. B. A. Whitelaw und D. F. Carlson. „95 PRODUCTION OF GENE-EDITED PIGS, CATTLE, AND LAMBS BY EMBRYO INJECTION OF TALENS OR ZFNs“. Reproduction, Fertility and Development 26, Nr. 1 (2014): 161. http://dx.doi.org/10.1071/rdv26n1ab95.
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Transcription activator-like effector nuclease (TALEN) and zinc finger nuclease (ZFN) DNA editing technology enables site-directed engineering of the genome. To date, all gene-edited large animals have been produced by treatment of somatic cells and cloning to produce gene-edited offspring. Although effective, it does not take advantage of the ‘leave-no-trace' aspect of site-specific nucleases, and the derivation of food animals by cloning is negatively perceived by the public. Thus, we have investigated production of gene-edited pigs, cattle and sheep by direct injection of TALEN or ZFN mRNAs to develop loss-of-function alleles for disease resistance (RELA) or enhanced meat production (GDF8). In vitro studies demonstrated activity of TALENs by cytoplasmic injection of mRNAs from dosages of 2 to 20 ng mL–1 with an apparent increase in both editing frequency and toxicity at high dosage. Our first pregnancies were produced by transfer of pig embryos (in vivo produced) injected with 2 ng mL–1 RELA TALEN mRNA. Pregnancy was confirmed in 5 of 7 recipients 4 of which went full term giving rise to 39 piglets, 8 of which carried editing events (21%). In parallel, we injected ZFN mRNA (2 ng mL–1) targeted to a similar site of the RELA gene and 2 of 2 recipients became pregnant, resulting in the birth of 9 piglets, one of which was edited (11%). For cattle injections, we derived zygotes by ovum pickup from selected Nelore dams followed by in vitro maturation and fertilization with Nelore semen. Low (2 ng mL–1) and medium (5 ng mL–1) dosages of GDF8-targeted TALENs resulted in Day 7 development to morula/blastocyst stage in 40% (n = 18) and 10% (n = 66) of cultured embryos, respectively. A total of 20 morula/blastocysts were chosen for transfer to 11 recipients, resulting in two full-term pregnancies. One pregnancy produced two calves, both of which carried edited GDF8 alleles. Complications with parturition of the second pregnancy resulted in 2 stillborn calves, the genotypes of which are under investigation. Finally, 2 ng mL–1 of TALEN mRNA targeted to ovine GDF8 was injected into in vivo-produced sheep zygotes and transferred into nine recipients, 3 blastocysts each. The pregnancy rate, number of live-born animals, and gene editing frequency is under investigation and will be reported.
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Fahrenkrug, Scott C., Wenfang Tan, Simon G. Lillico, Dana Stverakova, Chris Proudfoot, Gayle Williamson, Charles R. Long, Bruce A. Whitelaw und Daniel F. Carlson. „GENE INACTIVATION AND NONMEIOTIC ALLELE INTROGRESSION IN LIVESTOCK SPECIES USING TALENS“. Reproduction, Fertility and Development 25, Nr. 1 (2013): 318. http://dx.doi.org/10.1071/rdv25n1ab340.
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TALEN-induced double-strand breaks can be used for gene inactivation via repair by non-homologous end joining (NHEJ) or to stimulate homologous recombination (HR). HR can be used to introduce custom genetic modifications or to introgress naturally occurring alleles. We found that over 65% of custom-designed TALENs displayed activity in pig and cattle fibroblasts, with a typical percentage of indel positive chromosomes ranging from 20 to 45%. Isolation of individual clones with mono- and biallelic modifications to targeted loci was extremely efficient (up to 84 and 24% of clones, respectively) and could be accomplished without the aid of selection. Co-transfection of TALENs with a homologous repair template enabled precise insertion of a novel restriction site in nearly 40% of treated cells, with surprising levels of homozygosity. To prove that gene-edited Ossabaw swine cells were suitable for the generation of animals by cloning, we pooled colonies harboring both monoallelic and biallelic TALEN-induced frame-shift mutations in the swine low-density lipoprotein receptor (LDLR) and used them as nuclear donors for chromatin transfer. Pregnancy was established in 7/9 transfers, and 6 pregnancies were carried to term, resulting in the live birth of 18 piglets. Pigs heterozygous and homozygous for TALEN-induced mutations are being investigated as models of familial hypercholesterolemia (FH). We have additionally targeted the same locus for HR using a specified inactivating mutation. Fibroblasts heterozygous and homozygous for a specific 4-bp insertion into LDLR were created by allele introgression and have been cloned by chromatin transfer, demonstrating that gene editing can be used to create precise, swine knock-ins in a single generation. Allele introgression is also critical to livestock genetics, where crossbreeding has been a staple of breeding programs. Although major effect alleles for enhancing productivity and animal welfare have been discovered, the introgression of low-frequency alleles by traditional breeding is slow and inaccurate, involving recombination across the entire genome. The development of gene editing technologies would provide the opportunity to accelerate the genetic improvement in a diversity of livestock breeds. Co-transfection of a TALEN pair with a template containing a specific, naturally occurring allele was effective at the non-meiotic introgression of quantitative traits into the genome of cells from naïve cattle breeds, now being used to create founders by cloning. We will also present progress towards gene conversion by direct injection of livestock embryos. Injection of TALEN mRNA into the cytoplasm of pig and cattle zygotes was capable of inducing gene knockout (KO) in up to 75% of embryos analysed, nearly half of which harbored biallelic modification. We will present alternative strategies for the incorporation of gene editing into livestock genetic improvement programs by either cloning or embryo treatment.
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Savy, V., R. J. Bevacqua, N. G. Canel, V. Alberio, L. D. Ratner, D. F. Carlson, M. I. Gismondi et al. „202 Combination of transcription activator-like effector nucleases and homology-independent target integration strategy gene editing technologies for knock-in of recombinant human factor IX Under the β-casein native promoter in bovine IVF embryos“. Reproduction, Fertility and Development 31, Nr. 1 (2019): 226. http://dx.doi.org/10.1071/rdv31n1ab202.
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Precise DNA modification is a crucial approach for gene function elucidation, biomedical model development, and transgenic bioreactor generation. In livestock, its application was extremely challenging until the development of engineered nucleases such as zinc-finger nucleases, transcription activator-like effector nucleases (TALEN), and CRISPR/Cas9. Still, precise knock-in (KI) techniques remain inefficient. Recently, the homology-independent target integration (HITI) strategy was developed, allowing precise insertion of transgenes in mammalian cells in an easier fashion. The HITI technique allows site-specific gene insertion by means of cleavage of both the target sequence in the genome and the donor plasmid, followed by DNA repair by nonhomologous end joining. Here, we evaluated the use of TALENs to generate precise knockout (KO) alleles of the β-casein gene (CSN2) by creating small insertions or deletions, and precise insertion of recombinant human factor IX (rhFIX) under bovine CSN2 regulatory sequences, using HITI via cytoplasmic injection of bovine IVF zygotes. First, 2 TALEN pairs (Tn1 and Tn2) targeting exon 2 of bovine CSN2 were designed and their activity was confirmed by primary fibroblasts transfection followed by Surveyor assay at Day 3. Then, both TALEN pairs were evaluated for KO embryo generation by zygote cytoplasmic injection of in vitro-transcribed mRNA encoding for Tn1, Tn2, or a mix containing Tn1+Tn2, at 100ng μL−1. A non-injected control (NIC) was also included. Embryos were in vitro cultured until Day 7 and independently analysed by whole-genome amplification followed by PCR and sequencing. Neither the blastocyst rate [28.8% (n=73), 33.8% (n=71), 32.4% (n=74), and 54.3% (n=127) for Tn1, Tn2, Tn1+Tn2, and NIC, respectively] nor the proportion of edited embryos [44% (n=9), 20% (n=10), and 33% (n=9) for Tn1, Tn2, and Tn1+Tn2, respectively] differed between injected groups (Fisher test, P<0.05), demonstrating efficient editing in bovine embryos by TALENs. Finally, to achieve precise CSN2 KI embryos, the rhFIX open reading frame was PCR amplified with a forward primer containing the Tn1 recognition sequence to obtain the HITI donor and bovine IVF zygotes were co-injected with the Tn1 mRNA and the HITI donor. Embryos were in vitro cultured until Day 7 and individually analysed by nested PCR at both the 5′ and 3′ ends of HITI donor. The PCR-based results indicate HITI donor integration in 7% of embryos analysed (n=14). Sanger sequencing analysis is currently in progress to confirm site-specific integration of HITI and possible rearranged DNA integration in other embryos. To our knowledge, this is the first report on the use of TALEN and HITI for gene modification. Our results indicate that TALEN combined with HITI may constitute an easy strategy for precise production of pharmaceuticals in the milk of livestock.
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de Frutos, C., D. Webster, S. C. Fahrenkrug und D. F. Carlson. „240 PRECISE GENOME EDITING OF PDX1 BY DIRECT INJECTION OF TRANSCRIPTION ACTIVATOR-LIKE EFFECTOR NUCLEASES (TALENS) INTO PARTHENOGENETIC PIG EMBRYOS“. Reproduction, Fertility and Development 28, Nr. 2 (2016): 252. http://dx.doi.org/10.1071/rdv28n2ab240.
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Pancreatic and duodenal homeobox 1 (PDX1) is one of the transcription factors involved in pancreatic organogenesis and plays a critical role as an early lineage marker of pancreatic specification and β-cell differentiation. In mature pancreas, PDX1 regulates a large number of genes involved in maintaining β-cell identity and function. In mice and humans, its homozygous disruption results in pancreas agenesis, while heterozygous mutations have been associated with early-onset (MODY) and late-onset forms of Type II diabetes mellitus in humans. Knockout of the PDX1 gene in pigs may lead to the generation of an apancreatic phenotype, which in turn could allow the potential generation of an exogenic pancreas. Moreover, this could help to create a large animal model for human diabetes. We used transcription activator-like effector nucleases (TALEN) technology with the aim of studying the efficiency of precise editing by homologous recombination in parthenogenetically activated porcine embryos. Mature oocytes were activated by incubation in ionomycin (10 µM) for 20 min, followed by a 4-h incubation in DMAP (2 mM) + cytochalasin B (7.5 µg mL–1). Cytoplasmic injection was performed 14 h post-activation. Post injections, embryos were cultured for 6 days in NCSU23+BSA media at 38.5°C in a 5% CO2 atmosphere. We first evaluated the activity of a pair of TALENs targeting the functional domain of the PDX1 gene. Three concentrations of mRNA were microinjected (10, 20, and 40 ng µL–1) and blastocysts were analysed for non-homologous end-joining (NHEJ) by sequencing. The efficiency of indel mutations at the PDX1-target loci (either monoallelic or biallelic) was 34.5, 52.6, and 80.0% for the 10, 20, and 40 ng µL–1 concentrations, respectively. Next, we tested whether ssODNs (single-stranded oligodeoxynucleotide) coinjected with TALENs would permit precise homology direct repair (HDR) in porcine parthenotes. TALEN mRNA (40 ng µL–1) was coinjected with an ssODN donor template (50 ng µL–1) designed to incorporate a novel stop codon, HindIII restriction site, and a frame shift mutation. Cleavage and blastocyst rates were recorded at Days 2 and 6 of development, respectively, in the TALEN/ssODN injected group (n = 260 zygotes), buffer-injected embryos (n = 135 zygotes), and the non-injected group (n = 132 zygotes). Day 7 embryos were analysed for NHEJ and HDR by RFLP assay and Sanger sequencing after whole-genome amplification and PCR. Blastocyst rates were 15% (TALEN/ssODN-injected group), 27% (buffer-injected group), and 34% (non-injected group). A total of 30 blastocysts were analysed for HDR after whole-genome amplification. The majority of analysed blastocysts (28/30, 93%) were mutant. Among them, 10 (36%) incorporated the ssODN, from which 3 (30%) showed a KO genotype with a precise biallelic modification. We report here a highly efficient and precise TALEN-mediated gene knockout in swine embryos, which represents an alternative to cloning for phenotype evaluation of knockouts related to organogenesis.
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Lee, Sanghoon, Changhong Yin, Janet Ayello, Michael Genualdi, Carmella van de Ven und Mitchell S. Cairo. „Transcription Activator Like Effector Nucleases (TALENs) Mediated CD20 Gene Knockout Burkitt Lymphoma Model“. Blood 122, Nr. 21 (15.11.2013): 4883. http://dx.doi.org/10.1182/blood.v122.21.4883.4883.
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Abstract Background Burkitt lymphoma (BL) is an aggressive mature B-cell malignancy and is the most common non-Hodgkin lymphoma (NHL) in children and adolescents (Cairo et al., Blood, 2007; Miles/Cairo et al., BJHaem, 2012) and the survival of pediatric Burkitt lymphoma (PBL) has significantly improved over the past 30 years through the introduction of short and intensive multi-agent chemotherapeutic regimens (Cairo et al, JCO, 2012). Patients with PBL who relapsed or progressed have chemotherapy resistant disease and can rarely be salvaged after re-induction and retreated (Cairo et al., JCO, 2012; Miles/Cairo et al., BJHaem, 2012). CD20 is expressed in over 98% of PBL (Perkins/Cairo et al, Clin Adv Hematol Oncol,2003). Targeting CD20 with rituximab in combination with chemotherapy has been utilized successfully in PBL (Goldman/Cairo, Leukemia, 2013). Targeting CD20 with monoclonal antibodies has been observed to alter signaling pathways, including MAPK, PI3K/AKT and NF-κB, by changing the expression of regulators of apoptosis leading to cell death and/or sensitizing cells to the effects of cytotoxic chemotherapeutic agents (Barth/Cairo, BJH, 2013; Mathas et al., 2000, Can. Res.; Jazirehi et al., Can. Res., 2005). CD20 is uniformly expressed in mature B cells and has been considered an effective target for immunotherapy because CD20 is not only widely expressed in mature B-cell but plays a role in human B-cell proliferation (Tedder et al., Immunol. Today, 1999). However, the mechanism(s) and function of CD20 is still poorly understood in B-cells, especially in BL. Transcription Activator-Like Effector Nucleases (TALENs) technologies have been developed for precision targeted genome editing in a variety of organisms and stem cells for new experimental and therapeutic tools (Sander et al., Nat. Biotech., 2011; Miller et al., Nat. Biotech., 2011). Objectives We hypothesize that 1) TALEN technology is suitable for the modification of CD20 gene expression in B cells and 2) TALENs-mediated CD20 knockout model will be provide useful tool for understanding the mechanism and function or CD20 and the potential use of CD20 antibodies as therapeutic agents in BL. Methods CD20 TALENs were constructed based on REAL (restriction enzyme and ligation) assembly methods (Sander et al., Nat. Biotech, 2011; Lee/Cairo et al, ASH, 2012) for CD20 gene modification. CD20 TALEN pairs or combinations were transfected into 293 and Raji cells using lipofectamin reagent (Invitrogen) and Amaxa nucleofection kit, respectively. Genomic DNA and total RNA were isolated using genomic DNA extraction kit (Promega) and Trizol reagent (Invitrogen). Surveyor mutation detection assay (Transgenomic) was used for CD20 TALENs validities and confirmed by sequencing analysis (Genewiz). Quantitative RT-PCR was performed by CFX96 Real-time system (Bio-rad) using qScript™ cDNA Synthesis Kit (Quantas) and SsoFast™ EvaGreen® Supermix (Bio-rad). Western blotting was performed for measurement of protein level and statistical significance was determined by a one tailed paired Student t-test. Results Two pairs of functional CD20 TALENs (T1and T2) targeting endogenous CD20 gene generated and confirmed their validities with gene modification caused by non-homologous end joining (NHEJ) in TALENs mediated 293 [Figure 1]. There was significant reduction of CD20 mRNA expression in T1, T2 and combined T1/T2 (T12) transfected in Raji compared to empty vector transfected Raji (50%, p<0.003; <40%, p<0.004 and 70%, p<0.003, respectively). To isolate of CD20 knockout single clone, transiently T12 transfected Raji cells were seeded into 96 well plates and screened by PCR genotyping. CD20 stably knockout clones, CD20-T12-29-1 and CD20-T12-29-5 (+/-) showed a significant decrease of CD20 mRNA (90% reduction, p<0.004; 70% reduction, p<0.002, respectively). We also observed a significant reduction of CD20 protein level by western blotting in single clones, CD20-T12-29-1 (no detectable CD20), CD20-T12-29-5 (97% reduction, p<0.002), CD20-T12-20-16 (98% reduction, p<0.00002) and CD20-T12-29-18 (75% reduction, p<0.006). Conclusions We demonstrated that CD20-TALENs are useful tools for the modification of endogenous CD20 gene and TALEN mediated CD20 knockout model will be used for studying CD20 function in BL and the efficacy of second generation CD20 antibodies and antibody conjugates against CD20 in BL. Disclosures: No relevant conflicts of interest to declare.
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Bevacqua, R. J., D. Carlson, R. Fernandez-Martín, A. E. Gibbons, V. Savy, N. G. Canel, G. V. Landschoot et al. „199 Efficient Knock-out of Ovine β-Lactoglobulin (BLG) Gene and Knock-in of Recombinant Human Factor IX (rhFIX) Under BLG Native Regulatory Sequences in Somatic Cells and Zygotes Using TALEN Nuclease“. Reproduction, Fertility and Development 30, Nr. 1 (2018): 240. http://dx.doi.org/10.1071/rdv30n1ab199.
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Site-specific genetic engineering is a valuable tool for pharmaceutical research and development of biomedical models. Despite engineered nucleases allow targeted gene edition in a rather simple fashion; few reports are available so far on specific gene knock-in (KI) combined with engineered nucleases in domestic species. Here, we evaluated the possibility of inducing specific KI of cDNAs coding for proteins of pharmaceutical interest under the control of milk native promoter sequences, taking advantage of the TALEN system, both in ovine somatic cells and in zygotes. We designed 2 TALENs, targeting exons 1 and 5 of ovine β-lactoglobulin gene (BLG), respectively, and a homologous recombination vector (pHR), carrying recombinant human factor IX (rhFIX) flanked by homology arms contiguous to the TALEN target sites. In an initial set of experiments, 5 × 105 to 1 × 106 ovine fibroblasts were transfected with 1 μg of each TALEN mRNA, with or without 50 ng μL−1 pHR. The feasibility of inducing knock-out (KO) was confirmed by Cel1 assay. The deletion of the genomic region between TALEN target sites and the occurrence of HR in cell lysates were assessed by PCR. Also, 576 individual colonies were picked up and analyzed by PCR. The deletion of the region between TALEN target sites was achieved with 7.8% efficiency (45/576). The incidence of HR in cells was 0.5% (3/576), as detected by PCR. In order to evaluate the system in zygotes, laparoscopic AI was performed on synchronized and superovulated ewes. Zygotes were recovered 16 h after AI and cytoplasmically injected with (1) 5 ng μL−1 TALEN mix (2.5 ng μL−1 oaBLG T1.1 + 2.5 ng μL−1 oaBLG T5.1) (5TM); (2) 5 ng μL−1 TALEN mix + 25 ng μL−1 pHR-hFIX plasmid (5TM+25pRH); or (3) 15 ng μL−1 TALEN mix (7.5 ng μL−1 oaBLG T1.1 + 7.5 ng μL−1 oaBLG T5.1) + 50 ng μL−1 pHR-hFIX (15TM+50pRH). A non-injected control (NIC) was also included. Embryo analysis was conducted on whole-genome amplified DNA from blastocysts, followed by PCR and sequencing. Non-parametric Fisher test was applied to detect significant differences among treatments. Although blastocyst rates for NIC and 5TM did not statistically differ, 5TM+25pRH and 15TM+50pRH groups resulted in lower blastocysts rates than the NIC [P < 0.05; 13/17 (76%), 6/15 (40%), 4/15 (26%) and 2/14 (14%) for NIC, 5TM, 5TM+25pRH and 15TM+50pRH respectively]. It was possible to detect the PCR product compatible with deletion of the entire region among TALEN target sites in 6/6 blastocysts (100%) from the group 5TM, 3/4 blastocysts (75%) from the group 5TM+25pRH and 2/2 (100%) blastocysts from the group 15TM+50pRH. HR was detected in 1/2 (50%) blastocysts injected with 15TM+50pRH and in 1/4 (25%) blastocysts injected with 5TM+25pRH, by PCR and sequencing of the PCR products. Our results indicate that TALEN combined with homologous recombination constitutes a powerful platform for the production of proteins of pharmaceutical interest under native regulatory sequences in the milk of genetically modified animals.
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Benjamin, Ronald, Atoshi Banerjee, Xiaogang Wu, Corey Geurink, Lindsay Buczek, Danielle Eames, Sara G. Trimidal, Janice M. Pluth und Martin R. Schiller. „XRCC4 and MRE11 Roles and Transcriptional Response to Repair of TALEN-Induced Double-Strand DNA Breaks“. International Journal of Molecular Sciences 23, Nr. 2 (06.01.2022): 593. http://dx.doi.org/10.3390/ijms23020593.
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Double-strand breaks (DSB) are one of the most lethal forms of DNA damage that, if left unrepaired, can lead to genomic instability, cellular transformation, and cell death. In this work, we examined how repair of transcription activator-like effector nuclease (TALEN)-induced DNA damage was altered when knocking out, or inhibiting a function of, two DNA repair proteins, XRCC4 and MRE11, respectively. We developed a fluorescent reporter assay that uses TALENs to introduce DSB and detected repair by the presence of GFP fluorescence. We observed repair of TALEN-induced breaks in the XRCC4 knockout cells treated with mirin (a pharmacological inhibitor of MRE11 exonuclease activity), albeit with ~40% reduced efficiency compared to normal cells. Editing in the absence of XRCC4 or MRE11 exonuclease was robust, with little difference between the indel profiles amongst any of the groups. Reviewing the transcriptional profiles of the mirin-treated XRCC4 knockout cells showed 307 uniquely differentially expressed genes, a number far greater than for either of the other cell lines (the HeLa XRCC4 knockout sample had 83 genes, and the mirin-treated HeLa cells had 30 genes uniquely differentially expressed). Pathways unique to the XRCC4 knockout+mirin group included differential expression of p53 downstream pathways, and metabolic pathways indicating cell adaptation for energy regulation and stress response. In conclusion, our study showed that TALEN-induced DSBs are repaired, even when a key DSB repair protein or protein function is not operational, without a change in indel profiles. However, transcriptional profiles indicate the induction of unique cellular responses dependent upon the DNA repair protein(s) hampered.
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Vidyarthi, Sanjeev. „Talen: Neighborhood“. Journal of the American Planning Association 86, Nr. 3 (28.05.2020): 379–80. http://dx.doi.org/10.1080/01944363.2020.1759957.
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YILMAZ, Gözde, Bahadır AYAS und Uğur SAK. „An Investigation of the Threshold Hypothesis Using ASIS Intelligence Scale and Creative Imagination Cards“. Talent 10, Nr. 2 (11.01.2021): 143–61. http://dx.doi.org/10.46893/talent.831465.
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KÖPRÜ, Ferhat, und Bahadır AYAS. „An Investigation of the Criterion Validity of Anadolu Sak Intelligence Scale (ASIS): The Case of EPTS“. Talent 10, Nr. 2 (11.01.2021): 110–28. http://dx.doi.org/10.46893/talent.857308.
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KILIÇARSLAN, Saadet, Bilge BAL SEZEREL und Uğur SAK. „An Investigation of the Relationship Between Speed-Based Verbal Reasoning Subtest of Anadolu-Sak Intelligence Scale and Perceptual Speed Tests“. Talent 10, Nr. 2 (11.01.2021): 129–42. http://dx.doi.org/10.46893/talent.844308.
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KAYACAN, Gamze, N. Nazlı ATEŞGÖZ und Uğur SAK. „A Comparative Analysis of Psychometric Properties of Memory Tasks and Their Relationships with Higher-Order Thinking Skills: Recognition Versus Recall“. Talent 10, Nr. 2 (11.01.2021): 162–75. http://dx.doi.org/10.46893/talent.847543.
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KARABULUT, Ridvan, und Enver TÜRKSOY. „Perceptions of Gifted Students Towards Distance Education in the Covid-19 Pandemic“. Talent 10, Nr. 2 (11.01.2021): 176–89. http://dx.doi.org/10.46893/talent.773442.
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Haarmeyer, Hans. „Verfahren in Talaren“. return 4, Nr. 3 (September 2017): 70–71. http://dx.doi.org/10.1007/s41964-017-0092-3.
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Prajartoro, Albertus. „Seleksi Pemimpin High Potential Talent Dengan Strategi Parenting Program“. KILAT 9, Nr. 1 (25.04.2020): 85–91. http://dx.doi.org/10.33322/kilat.v9i1.846.
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PT PLN (Persero) has the potential of lacking leaders at the Top Managers and Middle Managers level in the next 10 years. It caused by a very rapid organizational development and delay in the regeneration due to zero growth in 1996-2001 and the existing programs have not been able to meet the needs. To anticipate this, Parenting Program is proposed to be a mentoring and counseling program for High Potential (HiPO) Talen to become a future PLN’s leader with good character and excellent performance. This Program is applied by selecting employees who are in the HiPO criteria and constructed through the parenting program. This study resulted in the determination of Hipo Talent criteria and the criteria of the parent, as well as the proposed program in the form of flow chart and roadmap implementation, and program materials.
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De Wilde, Truus. „Eeuwenlang talen leren“. Internationale Neerlandistiek 55, Nr. 3 (01.11.2017): 260–63. http://dx.doi.org/10.5117/in2017.3.wild.
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van Rijn, Inge. „De 100 talen“. Kinderopvang 33, Nr. 5 (31.07.2023): 8–10. http://dx.doi.org/10.1007/s41189-023-1957-y.
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Solís Figueroa, Raúl, Michele Berho Montalvo, Marion Koch und Francisca Rodríguez Leonard. „Cuerpo Taller: Aproximación metodológica al Taller de Introducción a la Arquitectura“. AUS, Nr. 18 (2015): 44–51. http://dx.doi.org/10.4206/aus.2015.n18-08.
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Wu, Haibo, Yongsheng Wang, Yan Zhang, Mingqi Yang, Jiaxing Lv, Jun Liu und Yong Zhang. „TALE nickase-mediatedSP110knockin endows cattle with increased resistance to tuberculosis“. Proceedings of the National Academy of Sciences 112, Nr. 13 (02.03.2015): E1530—E1539. http://dx.doi.org/10.1073/pnas.1421587112.
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Transcription activator-like effector nuclease (TALEN)-mediated genome modification has been applied successfully to create transgenic animals in various species, such as mouse, pig, and even monkey. However, transgenic cattle with gene knockin have yet to be created using TALENs. Here, we report site-specific knockin of the transcription activator-like effector (TALE) nickase-mediated SP110 nuclear body protein gene (SP110) via homologous recombination to produce tuberculosis-resistant cattle. In vitro and in vivo challenge and transmission experiments proved that the transgenic cattle are able to control the growth and multiplication ofMycobacterium bovis, turn on the apoptotic pathway of cell death instead of necrosis after infection, and efficiently resist the low dose ofM.bovistransmitted from tuberculous cattle in nature. In this study, we developed TALE nickases to modify the genome of Holstein–Friesian cattle, thereby engineering a heritable genome modification that facilitates resistance to tuberculosis.
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Wang, Ming, ZhaoLin Sun, Fangrong Ding, Haiping Wang, Ling Li, Xue Li, Xianjin Zheng, Ning Li, Yunping Dai und Changxin Wu. „Efficient TALEN-mediated gene knockin at the bovine Y chromosome and generation of a sex-reversal bovine“. Cellular and Molecular Life Sciences 78, Nr. 13 (28.05.2021): 5415–25. http://dx.doi.org/10.1007/s00018-021-03855-1.
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AbstractFunctional elucidation of bovine Y-chromosome genes requires available genome editing technologies. Meanwhile, it has yet to be proven whether the bovine Sry gene is the main or single factor involved in the development of the male phenotype in bovine. Here, we efficiently knocked out four Y-linked genes (Sry, ZFY, DDX3Y, and EIF2S3Y) in bovine fetal fibroblasts (BFFs) with transcription activator-like effector nucleases (TALENs) individually. Furthermore, we used TALEN-mediated gene knockin at the Sry gene and generated a sex-reversal bovine by somatic cell nuclear transfer (SCNT). The resulting bovine had only one ovary and was sterile. We demonstrate, for the first time, that the Sry gene is an important sex-determining gene in bovine. Our method lays a solid foundation for detecting the biology of the bovine Y chromosome, as it may provide an alternative biological model system for the study of mammalian sex determination, and new methods for the practical application in agricultural, especially for sex predetermination.
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Hess, Paul, Gregory Gojanovich und Jennifer Holmes. „Eradication of surface MHC class Ia expression in an insulinoma cell clone by TALEN-mediated gene deletion prevents recognition by diabetogenic CD8+ T cells (THER5P.843)“. Journal of Immunology 192, Nr. 1_Supplement (01.05.2014): 200.22. http://dx.doi.org/10.4049/jimmunol.192.supp.200.22.
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Abstract In type 1 diabetes, islet transplantation to replace β cells killed by pathogenic T cells is a promising means of attaining insulin independence; however, graft durability is limited by auto- & alloreactive responses. While autologous stem cells ultimately may circumvent the latter obstacle, the inciting CD8+ T-cell effectors are a threat to long-term engraftment. Islet cell manipulation to prevent expression of class Ia molecules could render them “invisible” to diabetogenic CTL, & we hypothesized that transcription activator-like effector nucleases (TALENs) could be used to engineer this modification. TALENs designed to flank a Bgl1 site in exon 1 of H2-Kd/Db were transfected into the NOD insulinoma line NIT-1, & cells with high nuclease activity were sorted using a GFP reporter plasmid encoding the target sequence. A clone isolated by limiting dilution (KG) was negative for H2-Kd/Db surface expression, & PCR amplicons were resistant to Bgl1 digestion. Genomic DNA sequencing showed biallelic modifications of both genes, with one Db sequence containing a 4 bp insertion. The remaining 3 alleles appeared to be repaired by gene conversion, causing frameshift mutations. When pulsed with cognate peptide, KG failed to stimulate diabetogenic CD8+ T cells purified from TCR-transgenic NOD.8.3 mice, as measured by CFSE dilution & IFN-γ production. These data demonstrate that TALEN-mediated genomic modification of insulin-producing cells can prevent recognition by autoreactive CTL.
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Cornips, Leonie. „Taalcultuur: Talen in beweging“. Taal en tongval 65, Nr. 2 (01.07.2013): 125–47. http://dx.doi.org/10.5117/tet2013.2.corn.
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Sørensen, Anders Dræby. „Talen begynder efter døden:“. Slagmark - Tidsskrift for idéhistorie, Nr. 71 (18.08.2015): 266–69. http://dx.doi.org/10.7146/sl.v0i71.107324.
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Bratt, Rachel G. „Neighborhood, by Emily Talen“. Journal of Urban Affairs 42, Nr. 3 (22.07.2019): 474–76. http://dx.doi.org/10.1080/07352166.2019.1638181.
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van Bemmel, Marlies, Jan s. Jukema und Nicole Ketelaar. „Eén praktijk, twee talen“. Denkbeeld 28, Nr. 4 (August 2016): 6–9. http://dx.doi.org/10.1007/s12428-016-0052-4.
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Paffen, Peter. „Spreektoetsen Moderne Vreemde Talen“. Spreken in moedertaal en vreemde taal 54 (01.01.1996): 145–52. http://dx.doi.org/10.1075/ttwia.54.14paf.
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In 1988 CITO started research into the feasibility of valid and reliable oral proficiency tests based on communicative principles. This was to meet the demand for a communicative speech test to be used in school based examinations in secondary education. Using the Test of Spoken English as a guideline, tests for French, German and English were developed. Simultaneous research into the reliability and validity of the tests led to various adaptations of the original model. From 1992 onwards oral proficiency tests for each of the three languages in question have been published at levels VBO/MAVO, HAVO and VWO (approxi-mately: vocational, secondary modern and grammar school). The results of a user inquiry held in 1994 led to a number of further changes to improve the user-friend-liness of the tests. Early in 1996 a new research project concerning the reliability and validity of the tests was started. The results will be published in the autumn of 1996.
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Nienhuis, Lydius, Wim Danhof und Marijke Peet. „Leessnelheid in Vreemde Talen“. Toegepaste Taalwetenschap in Artikelen 63 (01.01.2000): 33–40. http://dx.doi.org/10.1075/ttwia.63.04nie.
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This paper presents the results of two studies on the reading rate of students after 5 years of secondary education, including French and English as foreign languages. The reading processes investigated were, in the terminology of Carver, rauding (i.e. 'normal' reading), scanning and learning. As is known from research by Segalowitz, even skilled bilinguals attain an L2 reading rate of only 60-70% of that in their native language. Our students cannot be considered really 'skilled' bilinguals. So it is not surprising that, during the 'normal' reading process (i.e. the process of rauding) of French texts, our students arrive at a reading rate that is considerably slower; they attain a reading rate of 42-48% of the rate of comparable Tl readers; they read about 104 words a minute in French. The reading rate of our students in English as a foreign language is somewhat better: they arrive at a rate of 137 words a minute; this corresponds to 58.6% of the reading rate of comparable Tl readers in English. Strikingly, reading rates in three reading processes: scanning, rauding and learning, as described by Carver, differed proportionally for our readers of French in nearly the same way as for native-language readers of English. In the three cases, students arrived at a reading rate of about 40-45% of that in native-language reading.
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Broeder, Peter. „Talen Leren in Europa“. Toegepaste Taalwetenschap in Artikelen 65 (01.01.2001): 97–109. http://dx.doi.org/10.1075/ttwia.65.10bro.
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From 1989 until 1997 the project Language Learning/or European Citizenship was carried out under the auspices of the Council of Europe in Strasbourg. In this project two instruments were developed. The first instrument is the European Framework of Reference. This is a kind of language scale through which knowledge and qualifications in different languages can be compared. The second instrument is the European Language Portfolio. This is a kind of language passport through which languages users can report and document their proficiency in, and use of different languages. This contribution gives a description of these instruments. In addition, there is a summary of the results of a pilot project in the Netherlands which focused on the multilingual classroom at the end of primary school.
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Gorter, Durk. „Meertaligheid en Bedreigde Talen“. Thema's en trends in de sociolinguistiek 4 70 (01.01.2003): 27–38. http://dx.doi.org/10.1075/ttwia.70.04gor.
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In this article, the point of departure is the contradictor}' developments of increased multilingualism in society and, at the same time, the danger of the disappearance of some languages. The case of Friesland is used to illustrate these processes. I European efforts to safeguard and protect languages are briefly discussed, and the framework of the Kuromosaic study is mentioned. The language situation in Friesland has always involved more than just the bilingualism of Frisian and Dutch. The number of languages in daily life has increased over the last few years, but dialects such as Town-Frisian or the dialects on the Wadden-islands are spoken less well by the next generation. These processes can clearly be demonstrated by the local changes in the town of Hindeloopen and the provincial capital Leeuwarden. In Hindeloopen, proficiency in the local Frisian dialect has been halved in 50 years. In the capital, over 50 different languages are spoken at home nowadays. In the 'linguistic landscape' (the public display of languages) Frisian occupies a minor place. English and Dutch are prominent. It is concluded that these trends will continue, and some suggestions for further research into these new phenomena of multilingualism are given.
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Berzsenyi, George. „Talent Search versus Talent Development“. Notices of the American Mathematical Society 66, Nr. 09 (01.10.2019): 1. http://dx.doi.org/10.1090/noti1950.
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Forner, Joachim, Dennis Kleinschmidt, Etienne H. Meyer, Axel Fischer, Robert Morbitzer, Thomas Lahaye, Mark A. Schöttler und Ralph Bock. „Targeted introduction of heritable point mutations into the plant mitochondrial genome“. Nature Plants 8, Nr. 3 (März 2022): 245–56. http://dx.doi.org/10.1038/s41477-022-01108-y.
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AbstractThe development of technologies for the genetic manipulation of mitochondrial genomes remains a major challenge. Here we report a method for the targeted introduction of mutations into plant mitochondrial DNA (mtDNA) that we refer to as transcription activator-like effector nuclease (TALEN) gene-drive mutagenesis (GDM), or TALEN-GDM. The method combines TALEN-induced site-specific cleavage of the mtDNA with selection for mutations that confer resistance to the TALEN cut. Applying TALEN-GDM to the tobacco mitochondrial nad9 gene, we isolated a large set of mutants carrying single amino acid substitutions in the Nad9 protein. The mutants could be purified to homochondriomy and stably inherited their edited mtDNA in the expected maternal fashion. TALEN-GDM induces both transitions and transversions, and can access most nucleotide positions within the TALEN binding site. Our work provides an efficient method for targeted mitochondrial genome editing that produces genetically stable, homochondriomic and fertile plants with specific point mutations in their mtDNA.
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טלמון, ישעיהו. „אנליזה ננו–מבנית של נוזלים מורכבים באמצעות מיקרוסקופיית אלקטרונים בטמפרטורה קריוגנית“. איגרת האקדמיה הלאומית הישראלית למדעים, Nr. 44 (20.12.2022): 20–27. http://dx.doi.org/10.52873/igeret.2022.44.talmon.
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Noh, Hee-Jung, Yoonsung Kang und Keun-Cheol Kim. „TALEN Constructs and Validation for Targeting of SETDB1 Genomic DNA“. Journal of Life Science 24, Nr. 12 (30.12.2014): 1269–75. http://dx.doi.org/10.5352/jls.2014.24.12.1269.
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Maas-Sundararaman, Deepa, und Esther Sifuma. „How Talent Acceleration Generated Talent Advocacy“. HemaSphere 2, Nr. 3 (Juni 2018): e46. http://dx.doi.org/10.1097/hs9.0000000000000046.
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Schiemann, William A. „From talent management to talent optimization“. Journal of World Business 49, Nr. 2 (April 2014): 281–88. http://dx.doi.org/10.1016/j.jwb.2013.11.012.
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Serban, Anca, und Marcela Andanut. „Talent Competitiveness and Competitiveness through Talent“. Procedia Economics and Finance 16 (2014): 506–11. http://dx.doi.org/10.1016/s2212-5671(14)00831-4.
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Skuza, Agnieszka, Habte G. Woldu und Shawn Alborz. „Who is talent? Implications of talent definitions for talent management practice“. Economics and Business Review 8 (22), Nr. 4 (2022): 136–62. http://dx.doi.org/10.18559/ebr.2022.4.7.
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Although talent is considered imperative for gaining a competitive advantage, talent management programs’ effectiveness is unknown. It is believed that consensus on a strong theoretical underpinning for identifying talent and its general definition is yet to be achieved among academia and practitioners. This lack of integration and agreement on a single definition among scholars lead to more confusion which inhibits the advancement of talent management scholarship. The notion also requires renewed attention in the post-pandemic era because everything may not go back to normal as pre-pandemic. This study addresses the gap and focuses on reviewing the existing scholarship on talent definitions and its conceptualization in one place. The study also aims to present the potential implications of talent definition on talent management practices. Among the various implications discussed, it is argued that a single approach to talent definition makes the company vulnerable as it is not using the full potential of talent management. Finally, based on this in-depth review, the study will highlight potential critical research areas towards which the scholarship of talent may be extended.
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K.S, Dr Usman Mohideen. „The Mediating Role of Organizational Commitment in Relationship between Talent Management Practices and Talent Retention“. International Journal of Research in Arts and Science 5, Special Issue (30.08.2019): 174–87. http://dx.doi.org/10.9756/bp2019.1002/16.
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Petersen, Björn. „UPDATE ON 'MOLECULAR SCISSORS' FOR TRANSGENIC FARM ANIMAL PRODUCTION“. Reproduction, Fertility and Development 25, Nr. 1 (2013): 317. http://dx.doi.org/10.1071/rdv25n1ab339.
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Molecular scissors, such as meganucleases, zinc-finger nucleases (ZFN), and transcription activator-like effector nucleases (TALEN), are valuable tools for generating double-strand breaks (DSB) in the genome that can lead to a functional knockout of the targeted gene or used to integrate a DNA sequence at a specific locus in the genome. Especially in farm animal species from which true pluripotent embryonic stem cells have not been established, these molecular scissors are a new option for engineering the genome in a way that was not feasible before. Meganucleases (also called homing nucleases) are natural proteins found in many single-cell organisms that are mainly involved in the cell’s repair mechanism after a strand break occurs. They are capable of recognising their binding site by identifying a sequence containing between 12 and >30 base pairs. The prototype enzyme for demonstrating DSB stimulation of gene targeting was I-SceI, which has a long recognition site (I-SceI 18 bp). The recognition specificity of enzymes such as I-SceI can be modified to be specific for a desired sequence within the genome. The use of meganucleases to genetically modify organisms has proved very successful in several species, including frog, fly, fish, plants, and human cells, but the intimate connection between the recognition and cleavage elements in the protein structure makes it difficult to alter one without affecting the other. The class of targeting reagents that has proved the most versatile and effective in recent years is that of ZFN. The ZFN possess separate DNA-binding and cleavage domains, which facilitate design according to the desired target. These molecules originate from the natural type IIS restriction enzyme FokI (Li et al. 1992 Proc. Natl. Acad. Sci. USA 89, 4275–4279). The cleavage domain has no sequence specificity and the binding domain can be used to make ZFN specific to a targeted sequence. The requirement for dimerisation of the FokI makes ZFN even more specific and avoids off-target events, as a monomeric cleavage does not occur at single binding sites. One zinc-finger molecule is specific for a base triplet; joining several zinc-finger molecules is sufficient to pick out a single target in a complex genome. ZFN have been used to modify the genome of several species as Xenopus, drosophila, C. elegans, zebrafish, rat, mouse, human cells, hamster cells, rabbit, pigs, and cattle. Different methods have been used to alter the host genomes either by ZFN mRNA or DNA injection into zygotes or by transfection of somatic cells followed by somatic cell nuclear transfer. Even a direct delivery of ZFN proteins can generate a targeted mutation (Gaj et al. 2012 Nat. Methods 9, 805–807). The efficiency of ZFN-mediated knockout was increased up to 10,000-fold compared with traditional gene knockout by homologous recombination. Rarely, off-target events were described but most were located in an intergenic or intronic region of the genome. Transcription activator-like effectors are a family of virulence factors produced by a genus of plant pathogens, Xanthomonas spp. The proteins naturally comprise 17 to 18 repeats of 34 amino acids. The binding specificity is determined by the amino acids at positions 12 and 13 within each repeat. Combined with an endonuclease, TALEs (referred to as TALENs) can be used to specifically target almost any known genomic sequence. The main difference between ZFNs and TALENs is the recognition of the DNA sequence. While ZFNs recognise nucleotide triplets, TALENs recognise single nucleotides, rendering TALENs, in theory, adjustable to any given sequence in a genome while ZFNs need defined prerequisites to be specific. TALENs have already been used to alter the genomes of rats, zebrafish, human iPSCs, and pigs (personal communication). Molecular scissors open a wide range of new applications for modifying the genome of different species or cells with which it has remained very difficult to work. Breeding for agricultural purposes and biomedicine, including the development of large animal models for human diseases and xenotransplantation, will greatly benefit from these new tools. With the advent of ZFN- and TALEN-mediated gene knockouts, mammalian transgenesis has taken a major leap forward as a straightforward technology for gene knockout and knock-in.