DNA templates for in vitro transcription were generated by PCR amplification of plasmids encoding the EBOV 3E-5E-GFP minigenome and the corresponding replicon mutants, using primers listed in Table 1 . All PCR experiments were performed using Platinum® Taq DNA polymerase High Fidelity (Thermo Fisher Scientific, Waltham, MA, USA). Transcripts were synthesized with the T7-MEGAscript system (Thermo Fisher Scientific, Waltham, MA, USA) following the manufacturer's protocol. RNAs were purified by denaturing 8 M urea/5% polyacrylamide gel electrophoresis, followed by elution and ethanol precipitation. Purified RNAs were dissolved in sterile water and stored at −20°C.
T7 megascript system
Manufactured by Thermo Fisher Scientific
Sourced in United States
The T7 MEGAscript system is a high-yield in vitro transcription kit designed for the synthesis of large amounts of RNA from DNA templates. The kit utilizes the T7 RNA polymerase enzyme to transcribe DNA sequences into RNA, enabling the production of various RNA species for a wide range of applications.
Automatically generated - may contain errors
Lab products found in correlation
Platinum taq dna polymerase high fidelity, by Thermo Fisher Scientific (1 mentions)
Megaclear transcription clean up kit, by Thermo Fisher Scientific (1 mentions)
Agilent bioanalyzer 2100 system, by Agilent Technologies (1 mentions)
Poly a polymerase tailing kit, by Illumina (1 mentions)
Turbo dnase 1, by Thermo Fisher Scientific (1 mentions)
Nd 1000, by Thermo Fisher Scientific (1 mentions)
6 protocols using t7 megascript system
1
In Vitro Transcription Protocol for EBOV Minigenome
2
KSHV PAN RNA Purification and Polyadenylation
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The pcDNA3.1 plasmid containing KSHV PAN RNA sequence was kindly provided by Joseph Ziegelbauer (NIH, HIV and AIDS Malignancy Branch). Transcription templates were amplified by PCR using primers listed in Table S1, and RNAs synthesized using the T7-MEGAscript system (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer's protocol. RNAs were fractionated over a denaturing 8 M urea/6% polyacrylamide gel, and purified by excision followed by electroelution and ethanol precipitation. Purified RNAs were dissolved in sterile water and stored at −20°C. PAN RNA was polyadenylated using Poly(A) Polymerase Tailing Kit (Epicentre) per manufacturer's protocol. Polyadenylated PAN RNA was purified using MEGAclear Transcription clean-up kit per manufacturer's instruction (ThermoFisher). The efficiency of poly(A) tailing was assessed using the Agilent 2100 Bioanalyzer system.
3
Disrupting CSFV E2-SERTAD1 Interactions
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A full-length Infectious Clone of the virulent CSFV Brescia strain (pBIC) [5 (link)] was used as a template to introduce E2 amino acid substitutions disrupting E2-SERTAD1 protein-protein interactions. These amino acid substitutions were identical to those mapped by the reverse yeast two-hybrid methodology. Residue substitutions T149A, Y325H and H335R were introduced into the native E2 sequence in the designed pBICΔSERTAD1 construct. The pBICΔSERTAD1 plasmid was obtained by DNA synthesis (Epoch Life Sciences, Sugar Land, TX, USA).
The CSFV pBICΔSERTAD1 full-length genomic clone was linearized with SrfI and in vitro transcribed using the T7 MEGAscript system (Ambion, Austin, TX). RNA was precipitated with LiCl and transfected into SK6 cells by electroporation at 500 volts, 720 ohms and 100 watts with a BTX 630 electroporator (BTX, San Diego, CA, USA). Cells were seeded in 6-well plates and incubated for 4 days at 37 °C and 5% CO2. Virus was detected by immunoperoxidase staining as described above, and stocks of rescued viruses were stored at ≤−70 °C. The full-length genome of the in vitro rescued CSFV E2ΔSERTAD1 virus (E2ΔSERTAD1v) was completely sequenced by NGS.
The CSFV pBICΔSERTAD1 full-length genomic clone was linearized with SrfI and in vitro transcribed using the T7 MEGAscript system (Ambion, Austin, TX). RNA was precipitated with LiCl and transfected into SK6 cells by electroporation at 500 volts, 720 ohms and 100 watts with a BTX 630 electroporator (BTX, San Diego, CA, USA). Cells were seeded in 6-well plates and incubated for 4 days at 37 °C and 5% CO2. Virus was detected by immunoperoxidase staining as described above, and stocks of rescued viruses were stored at ≤−70 °C. The full-length genome of the in vitro rescued CSFV E2ΔSERTAD1 virus (E2ΔSERTAD1v) was completely sequenced by NGS.
4
Engineering Virulent CSFV Strain Variant
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A full-length infectious clone (IC) containing the complete genome of the virulent CSFV Brescia strain (pBIC) [5 (link)] was used as a template to incorporate the amino acid substitutions in the E2 gene to disrupt the E2-DOCK7 interaction. Residue substitutions Y65F V283D and T149A were introduced into the native E2 sequence to obtain the E2ΔDOCK7 construct. The E2ΔDOCK7 plasmid was obtained by DNA synthesis (Epoch Life Sciences, Sugar Land, TX, USA).
The IC harboring the E2ΔDOCK7 full-length genome was transcribed using the T7 MEGAscript system (Ambion, Austin, TX, USA). RNA was precipitated and electroporated into SK6 cells as previously described [14 (link)]. Recombinant virus E2ΔDOCK7 was then harvested and stocks were kept at −70 °C until use.
The IC harboring the E2ΔDOCK7 full-length genome was transcribed using the T7 MEGAscript system (Ambion, Austin, TX, USA). RNA was precipitated and electroporated into SK6 cells as previously described [14 (link)]. Recombinant virus E2ΔDOCK7 was then harvested and stocks were kept at −70 °C until use.
5
Generation of CSFV E2ΔCCDC115 Mutant
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A full-length IC of the virulent CSFV Brescia strain (pBIC) [4 (link)] was used as a template in which E2 amino acid substitutions disrupting the E2–CCDC115 interaction, as mapped by reverse yeast two-hybrid methodology, were included. Residue substitutions, K112M, N260I, and M275S, were introduced into the native E2 sequence in the designed pBICΔCCDC115 construct. The pBICΔCCDC115 plasmid was obtained by DNA synthesis (Epoch Life Sciences, Sugar Land, TX, USA).
The CSFV pBICΔCCDC115 full-length genomic clone was linearized with SrfI and in vitro transcribed using the T7 MEGAscript system (Ambion, Austin, TX, USA). RNA was precipitated with LiCl and transfected into SK6 cells by electroporation at 500 volts, 720 ohms, and 100 watts, with a BTX 630 electroporator (BTX, San Diego, CA, USA). Cells were seeded in 12-well plates and incubated for 4 days at 37 °C and 5% CO2. Virus was detected by immunoperoxidase staining as described above, and stocks of rescued viruses were stored at ≤ −70 °C. Full-length genome of in vitro rescued CSFV E2ΔCCDC115 virus (E2ΔCCDC115v) was completely sequenced by NGS.
The CSFV pBICΔCCDC115 full-length genomic clone was linearized with SrfI and in vitro transcribed using the T7 MEGAscript system (Ambion, Austin, TX, USA). RNA was precipitated with LiCl and transfected into SK6 cells by electroporation at 500 volts, 720 ohms, and 100 watts, with a BTX 630 electroporator (BTX, San Diego, CA, USA). Cells were seeded in 12-well plates and incubated for 4 days at 37 °C and 5% CO2. Virus was detected by immunoperoxidase staining as described above, and stocks of rescued viruses were stored at ≤ −70 °C. Full-length genome of in vitro rescued CSFV E2ΔCCDC115 virus (E2ΔCCDC115v) was completely sequenced by NGS.
6
Site-Directed Mutagenesis of mAb-2B6 Epitope
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Plasmid pA-RecC, a full-length cDNA clone of the C-strain viral genome, was used as the template in which each residue of the mAb-2B6 epitope was mutated by the Quick Change XL Site-Directed Mutagenesis Kit (Stratagene, USA). Each of the fulllength genomic clones was linearized by XhoI and transcribed in vitro using the T7 Megascript system (Ambion, Austin, USA). After TURBO DNase I (Ambion, USA) digestion, the RNA transcripts were precipitated with LiCl and quantified by a spectrophotometer, ND-1000 (Nano Drop Technologies, USA). The RNA transcripts were individually transfected into ST cells by electroporation at 150 V with a BTX 630 electroporator (BTX, San Diego, USA). Cells were plated in 25 cm 2 flasks and incubated for 4 days at 37°C and 5% CO 2 . Virus particles were detected by immunostaining the E2 protein as described below. Stocks of rescued viruses were stored at -80°C.
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