pMFG-OVA257–264-Cerulean, pMFG-GP61–80-Cerulean, and pMFG-OVA257–264-GP61–80-Ceruelan plasmids were constructed by inserting annealed oligonucleotides encoding triple SIINFEKL-AAY repeats, GLKGPDIYKGVYQFKSVEFD, or (SIINFEKL-AAY)3-P2A-GLKGPDIYKGVYQFKSVEFD, respectively, into the NcoI-linearized pMFG-Cerulean vector, as previously described [45 (link)]. Restriction enzymes were purchased from New England Biolabs. All constructs were verified by sequence analysis. Phoenix packaging cells (ATCC) were transfected with pMFG constructs; supernatants were used to transduce B16-F10 mouse melanoma tumor cell line to generate B16-F10 OVA257–264-Cerulean, B16-F10GP61–80-Cerulean and B16-F10 OVA257–264-GP61–80-Cerulean, respectively [45 (link)]. Transduced bulk cell lines were sorted for similar Cerulean expression levels.
Protocol detail
Engineered Melanoma Cell Lines for Vaccine Evaluation
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B16 melanoma
Bulk
Cell lines
Cells
Mouse
Oligonucleotides
Plasmids
Restriction enzymes
Sequence analysis
Tumor cell line
Vector
Corresponding Organization : Cornell University
Other organizations : Albert Einstein College of Medicine, Baylor College of Medicine
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Variable analysis
independent variables
- PMFG-OVA257–264-Cerulean plasmid
- PMFG-GP61–80-Cerulean plasmid
- PMFG-OVA257–264-GP61–80-Cerulean plasmid
- B16-F10 OVA257–264-Cerulean cell line
- B16-F10GP61–80-Cerulean cell line
- B16-F10 OVA257–264-GP61–80-Cerulean cell line
- Cerulean expression levels in transduced bulk cell lines
- Positive control: Not explicitly mentioned.
- Negative control: Not explicitly mentioned.
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