Transcript Construction of Reporter Luciferase Genes to Assess NOC4

Construction of Reporter
Luciferase Genes to Assess
NOC4 expression
Finding the Promoter Region of the
NOC4 Gene.
Nicholas Simon
Faculty Sponsor: Nancy Bachman
Overview
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Luciferase Vectors
Mutagenesis Strategy
Bioinformatics
Mutagenesis
Transformation
DNA Sequencing
Reporter Assays
Luciferase Vectors
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Vectors have four features
they are able to replicate
 they have selectable markers
 foreign DNA can be inserted in them
 they often carry a reporter gene
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Luciferase Vectors
Mutagenesis Strategy
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Point Mutations or Deletions?
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Point mutation in a region of 250bp –
need 30 primers
Deletion in a region of 250bp – need
only 6 primers
Issues: Cost and Saturation
Deletion was Best
Deletions
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cttctctatcgataggtaccgagctcttacgcgtaggtaccgagctcttacgcgtg
cggccgcgggctggcgggggacccttcaggcccggccccgtttgggcctcggct
cctggaaaagcgactcgcgcctctgggaagccgcagccccagactccagtcgc
gcttctcgcccggcgccgccggaaagcagcctctccaacgcctgccggaaagc
agcccggcccggcattttacgacgttcgcagcgctacccttttccgctccacggtg
acctccgtgcggccgggtgcgggcggagtcttcctcgatcccgtggtgctccgcg
gcgcggccttgctctcttccgggggctcgagatctgcgatctaagtaagcttggc
att
tttctctatcgataggtaccgagctcttacgcgtaggtaccgagctcttacgcgtg
cggccgcgggctggcgggggacccttcaggcccggccccgtttgggcctcggct
cctggaaaagcgactcgcgcctctgggaagccgcagccccagactccagtcgc
gcttctcgcccggcgccgccggaaagcagcctctccaacgcctgccggaaagc
agcccggcccggcattttacgacgttcgcagcgctacccttttccgctccacggtg
acctccgtgcggccgggtgcgggcggagtcttcctcgatcccgtggtgctccgcg
gcgcggccttgctctcttccgggggctcgagatctgcgatctaagtaagcttggc
att
General Strategy
Bioinformatics
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Used to design primers
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Minimize secondary structures
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Denature the DNA
template
Anneal the primers
Synthesize the
mutant strand
DNA Sequencing
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Purify the Vector
DNA from E. coli
Cycle DNA
sequencing
Reporter Assays
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Transfect the DNA vector into HeLa
cells
Wait for 24 hours so cell will
express the luciferase
Then measure luciferase activity
And What Happens If No Expression?