Journal: Virology

Article Title: Identification of two auto-cleavage products of nonstructural protein 1 (nsp1) in porcine reproductive and respiratory syndrome virus infected cells: nsp1 function as interferon antagonist

doi: 10.1016/j.virol.2009.11.033

Figure Lengend Snippet: Comparative sequence analysis of the nsp1α/nsp1β and nsp1β/nsp2 cleavage sites. Partial ORF1a amino acid sequence of six PRRSV strains corresponding to SD23983 ORF1a amino acid position 160–204 and 361–409 was aligned with EAV nsp1. The alignment was generated by ALIGNX program of Vector NTI Suite software (InforMax, Inc.). Two boxes depicted in the map of amino acid sequence comparison represent the result of nsp1β and nsp2 protein N-terminal sequencing, which was determined by sequential Edman degradation of 10 cycles for each viral protein. The upward and downward solid arrows point to the identified cleavage site of nsp1β/nsp2 and nsp1α/nsp1β, respectively. The downward dashed arrow points to the predicted nsp1α/nsp1β cleavage site.

Article Snippet: The alignment was generated by ALIGNX program of Vector NTI Suite software (InforMax, Inc.).

Techniques: Sequencing, Generated, Plasmid Preparation, Software

Journal: BMC Genomics

Article Title: A modified TALEN-based system for robust generation of knock-out human pluripotent stem cell lines and disease models

doi: 10.1186/1471-2164-14-773

Figure Lengend Snippet: Pretesting of TALENs and mutational spectra in 293 T cells and hPSCs. A : Illustration of GoldenGate TALEN assembly by Cermak et al. and cloning into new expression vectors pTAL7A and pTALB. Note that the number of RVDs is not limited to 18 but flexible. B : Schematic of negative selection of HPRT1 knock-out cells by 6-thioguanine (6-TG). 6-TG results in death of cells with functional HPRT1 . C : Cel-1 assay following transfection with three TALEN pairs (#1 to #3). Arrows indicate expected fragment sizes following Cel-1 digest of reannealed HPRT1 genomic PCRs (gPCR) as a result of error-prone NHEJ. Control plasmid (GFP) transfected cells served as negative control. Percentage of non-homologous end-joining (NHEJ) is indicated below each lane. D : Surviving 293 T cells following TALEN transfection and 6-TG selection of HPRT1 mutants. E : TALEN-induced mutation spectrum. Underlined wild-type sequence indicates binding region of TALEN pair #2. Mutant sequences shown are individual gPCR clones from bulk 6-TG selected cultures following transfection of TALEN pair #2. F : Pretesting of TALEN pairs #1 to #3 in HuES6 hESCs. Note that mutation efficiencies are somewhat decreased compared to 293 T cells in part B. G : Bulk hESC cultures following TALEN #2 transfection and 6-TG selection. H : TALEN-induced mutation spectrum in hESCs. All transfected cells were enriched by transient puromycin/blasticidin administration.

Article Snippet: These data demonstrate the universal applicability of the combined GoldenGate/pTAL7 TALEN system for generating knock-out hESC lines without the need for additional gene targeting vectors or negative selection procedures.

Techniques: TALENs, Clone Assay, Expressing, Selection, Knock-Out, Functional Assay, Transfection, Plasmid Preparation, Negative Control, Non-Homologous End Joining, Mutagenesis, Sequencing, Binding Assay

Journal: BMC Genomics

Article Title: A modified TALEN-based system for robust generation of knock-out human pluripotent stem cell lines and disease models

doi: 10.1186/1471-2164-14-773

Figure Lengend Snippet: Improved mutagenesis and universal applicability of the pTAL7 vector system. A : Cel-1 assay following transfection of 293 T and hESCs with HPRT1 talen pair #2 in different expression vector backbones. B : Functional confirmation of results in (A) by 6-TG selection. C : Quantification of functional mutations in HPRT1 following 6-TG selection, based on scoring of colony numbers. For the control without antibiotics and the pTAL4 vectors, no puromycin/blasticidin selection was performed. Note the strong increase in mutation rates upon pre-selection for double-transfected cells using transient administration of antibiotics. Error bars: SEM (n = 4). D : Applicability of the pTAL7 system to alternative genomic loci as shown by Cel-1 assay (left panel) or restriction digestion (right panel: NHEJ is indicated by enrichment of undigested fragments). hESCs were transfected with either a GFP control vector or the respective TALEN vectors, followed by subsequent gPCR amplification of TALEN target regions. E : Restriction digestion and characterization of 19 clones expanded after TALEN transfection without 6-TG selection. Except in panel C, pTAL7-transfected cells were routinely enriched by transient puromycin/blasticidin administration.

Article Snippet: These data demonstrate the universal applicability of the combined GoldenGate/pTAL7 TALEN system for generating knock-out hESC lines without the need for additional gene targeting vectors or negative selection procedures.

Techniques: Mutagenesis, Plasmid Preparation, Transfection, Expressing, Functional Assay, Selection, Amplification, Clone Assay

Journal: BMC Genomics

Article Title: A modified TALEN-based system for robust generation of knock-out human pluripotent stem cell lines and disease models

doi: 10.1186/1471-2164-14-773

Figure Lengend Snippet: Functional characterization of HPRT1 knock-out cell lines. A : HPRT1 lesions identified in 5 clonal hESC mutant lines. 10 PCR clones were sequenced per cell line. Wild-type sequences shown on top were not seen in any case suggesting that cell clones 1–3 are homozygous. Inserted DNA sequence of clone 5 is given in Additional file . B : Quantification of X chromosome copy number in clonal HPRT1 mutant lines using genomic qPCR for an X-linked locus, based on qPCR quantification. Note that all mutant clones cluster with the female parental control. C : Diagnostic PCR for detecting possible random integration of vector sequences into the genome of TALEN-treated cell clones. Analysis was performed at passage 5 after 6-TG selection. Original pTAL7 vectors served as positive control and isogenic parental cells served as negative control. D : PCR and subsequent Cel-1 assay for detection of mutations at the 5 most probably putative TALEN off-target cleavage sites in clonal HPRT1 knock-out cell lines. Genomic DNA from parental hESCs served as control. Note that the additional band in off-target site 1 probably results from a single nucleotide polymorphism and that there are no differences between clonal HPRT1 knock-out cell lines and the parental control. TALEN off-target binding sites are depicted in the right panel with mismatches indicated by asterisks.

Article Snippet: These data demonstrate the universal applicability of the combined GoldenGate/pTAL7 TALEN system for generating knock-out hESC lines without the need for additional gene targeting vectors or negative selection procedures.

Techniques: Functional Assay, Knock-Out, Mutagenesis, Clone Assay, Sequencing, Diagnostic Assay, Plasmid Preparation, Selection, Positive Control, Negative Control, Binding Assay

Journal: BMC Genomics

Article Title: A modified TALEN-based system for robust generation of knock-out human pluripotent stem cell lines and disease models

doi: 10.1186/1471-2164-14-773

Figure Lengend Snippet: Comparison of pTAL4 and pTAL7 vector systems

Article Snippet: These data demonstrate the universal applicability of the combined GoldenGate/pTAL7 TALEN system for generating knock-out hESC lines without the need for additional gene targeting vectors or negative selection procedures.

Techniques: Plasmid Preparation, Clone Assay, Expressing, Sequencing, Transfection, FLAG-tag