Rescue of Infectious Sindbis Virus by Yeast Spheroplast-Mammalian Cell Fusion
Abstract
:1. Introduction
2. Materials and Methods
2.1. Mammalian Cell Culture and Transfection
2.2. Yeast Cells and Growth Media
2.3. Plasmids
2.4. Yeast Transformation
2.5. Yeast Galactose Induction
2.6. Sindbis Virus
2.7. Yeast Spheroplasts Preparation and Storage
2.8. Yeast to BHK-21 Cell Fusion
2.9. Sindbis Virus Growth Curve and Titer Evaluation via Plaque Assay
2.10. Microscopy
3. Results
3.1. Yeast Centromeric Plasmids (YCps) for Galactose Induction in Saccharomyces Cerevisiae
3.2. Yeast Cytoplasmic Content Can Be Transferred to BHK-21 Cells via PEG Mediated Fusion
3.3. Infectious Sindbis Virus Is Produced via Yeast to BHK-21 Cell Fusion
4. Discussion
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Acknowledgments
Conflicts of Interest
References
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Plasmid Name | Purpose | Source |
---|---|---|
dsTE12Q-GFP | GFP tagged Sindbis genome (dsTE12Q) an infectious cDNA clone for in vitro transcription | A gift from Dr. Reed Shabman |
pLDJIF15 | Galactose inducible mRNA or protein, TRP1 (GenBank accession MW820849) | This study |
pLDJIF15-yeGFP | Galactose inducible yeGFP, TRP1 (GenBank accession MW82085) | This study |
pLDJIF19 | Galactose inducible mRNA or protein, HIS3 (GenBank accession MW820851) | This study |
pLDJIF20 | Galactose inducible mRNA or protein, URA3 (GenBank accession MW820852) | This study |
pLDJIF15-SINV | Galactose inducible dsTE12Q-GFP mRNA, TRP1 (GenBank accession MW820850) | This study |
Primer Name | Sequence 5′→3′ | Purpose |
---|---|---|
D66 | CAACCATAGGATGATAATGCGATTAG | Testing for insertion between Adapter 1 and 2 |
D67 | TGAGAAAGCAACCTGACCTACAG | |
D199 | ATTTCGATAAGCCAGTAAGCAGTGGGTTCT | Amplify pLDJIF15, 19, and 20 with adapter 1 and 2 |
D200 | CTGTGCCTTCTAGTTGCCAGCCATCTGTTG | |
D205 | AGAACCCACTGCTTACTGGCTTATCGAAATATGGTTAGTAAAGGTGAAGAATTATTCACT | Amplify yeGFP with adapter 1 and 2 |
D201 | CAACAGATGGCTGGCAACTAGAAGGCACAGTTATTTGTACAATTCATCCATACCATGGGT | |
D250 | aaaaaaaaaaaaaaaggggaattcctcgagCTGTGCCTTCTAGTTGCCAGCCATCTGTTG | Amplify pLDJIF15 backbone to insert SacI-SINV-GFP-XhoI cassette through Gibson Assembly |
D251 | gttctaacgacaatatgtccatacgagctcATTTCGATAAGCCAGTAAGCAGTGGGTTCT |
Name | Genotype | Source |
---|---|---|
W303α | MATα ade2-1 ura3-1 his3-11 trp1-1 leu2-3 leu2-112 can1-00 | ATCC® 208353™ |
yLDJIF9 | MATα ade2-1 ura3-1 his3-11 trp1-1 leu2-3 leu2-112 can1-00 [pLDJIF15] | This study |
yLDJIF21 | MATα ade2-1 ura3-1 his3-11 trp1-1 leu2-3 leu2-112 can1-00 [pLDJIF15-SINV] | This study |
yLDJIF22 | MATα ade2-1 ura3-1 his3-11 trp1-1 leu2-3 leu2-112 can1-00 [pLDJIF15-yeGFP] | This study |
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Ding, L.; Brown, D.M.; Glass, J.I. Rescue of Infectious Sindbis Virus by Yeast Spheroplast-Mammalian Cell Fusion. Viruses 2021, 13, 603. https://doi.org/10.3390/v13040603
Ding L, Brown DM, Glass JI. Rescue of Infectious Sindbis Virus by Yeast Spheroplast-Mammalian Cell Fusion. Viruses. 2021; 13(4):603. https://doi.org/10.3390/v13040603
Chicago/Turabian StyleDing, Lin, David M. Brown, and John I. Glass. 2021. "Rescue of Infectious Sindbis Virus by Yeast Spheroplast-Mammalian Cell Fusion" Viruses 13, no. 4: 603. https://doi.org/10.3390/v13040603