GeneArt™ CRISPR Nuclease Vector with CD4 Enrichment Kit

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	The presence of PEG and glycerol (supplied by the Ligation Buffer and the T4 DNA Ligase) will make the reaction mixture viscous. Be sure to mix the reaction thoroughly by pipetting up and down. Do not vortex.

Protocol tips
The presence of PEG and glycerol (supplied by the Ligation Buffer and the T4 DNA Ligase) will make the reaction mixture viscous. Be sure to mix the reaction thoroughly by pipetting up and down. Do not vortex.

Publication protocol

Plasmid constructs
The original plasmids containing the infectious cDNAs of the genomes of the following viruses: experimental vaccine strain of VEEV (VEEV TC-83, GenBank accession no. L01443), epizootic strain VEEV 3908 (GenBank accession no. U55350), laboratory strain SINV Toto1101 (SINV Toto1101, [57]), vaccine strain CHIKV (CHIKV-181/25, GenBank accession no. L37661), chimeric virus VEEV/CHIKV, which encodes VEEV TC-83 replication machinery and CHIKV LaReunion structural genes, EIL/5’TCVEEV-nLuc/VEEV and EIL/5’CHIKV-nLuc/VEEV were described elsewhere [58–63]. EIL/5’TCVEEV-nLuc/VEEV and EIL/5’CHIKV-nLuc/VEEV encode the replication machinery of the insect cell-specific Eilat alphavirus (EILV) and structural proteins of VEEV TC-83. The nLuc sequence was fused with VEEV TC-83 or CHIKV 5’UTRs and the amino-terminal fragment of the corresponding nsP1, and these cassettes were cloned under the control of an additional subgenomic promoter into the cDNA of the chimeric viral genome [29]. Other plasmids were designed using standard PCR-based techniques. The schematic representations of the modified genomes are shown in the corresponding figures. Plasmids, encoding alphavirus replicons VEErep/Flag-GFP-HVDsinv and SINrep/Flag-GFP-HVDsinv had the VEEV or SINV structural protein genes replaced by Flag-GFP, fused with HVD sequences derived from the indicated alphaviruses. The control replicons SINrep/Flag-GFP and VEErep/Flag-GFP encoded GFP, which was fused only with Flag. G3bp1, G3bp2, Frx1, Frx2 and Fmr1 genes were synthesized by RT-PCR using mRNA isolated from NIH 3T3 or BHK21 cells. These genes were cloned into modified PiggyBac plasmids (System Bioscience, Inc) under control of the CMV promoter. These plasmids were designed to express bi-cistronic mRNA, which encodes sequences of interest, and puromycin N-acetyltransferase (Pac) or blasticidin S resistance gene under the control of the EMCV IRES. Further modifications, such as fusions with GFP and deletions of the domain-coding sequences were introduced by PCR. Plasmids encoding the genomes of helper constructs, which were used for packaging of replicon RNAs into infectious viral particles, were described elsewhere [39, 64]. Sequences of the plasmids and details of the cloning procedures can be provided upon request.

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Discussion

Papers

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Manufacturer protocol

Download the product protocol from Thermo Fisher Scientific for GeneArt™ CRISPR Nuclease Vector with CD4 Enrichment Kit below.

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