Discuss limitations and advantages
• Tasmanian Devil
• Primary Cultures
• Mini Organ Cultures
• PLAP Assay - Reverse mutation detection assay, not forward. Mutant human
sequence was put inside the mouse (G11) and we are looking for the reverse
mutation back to G10. The mutation is a single base pair deletion.
• CII Assay - The mutation target is the CII gene. We sequenced about 400
nucleotides, however only 269 are the effective mutation target. Define mutation
target and effective mutation target (where you can detect the mutation). Lesser
number of sites where you can actually see the mutation.
• TCGA - a restriction site in RMC, this was the mutation target for Random
Mutation Capture. The mutation target and the effective mutation target are the
same. We are looking for it to be destructed, mutations can eliminate that
• Entire mitochondrial genome was both the mutation target and the effective
mutation target in the Ultra Deep Sequencing study.
• You could extend sequencing to the entire nuclear genome, however it is quite
• CII mutation detection assay - requires a special mouse (more expensive) and is
called the Big Blue Mouse. This mouse is transgenic (Tg) and it was
manufactured this way.
• Lambda is a virus and a bacteriophage. It infects E. Coli. Lambda DNA is about
50-55 kb in size and is double stranded DNA genome, also completely
sequenced. It is used to carry DNA into bacteria and we can use this to put DNA
in the mouse.
• In the making of the transgenic mouse, the company that made the mouse
would have many genomes. They can form concatomer (has many lambda
genes that are stuck end on end).
• The concatomer went into the mouse genome at chromosome four. These
chromosomes are telocentric from the MGDA paper. 40 copies in tandem of the
lambda genome on chromosome four and this is a mutation target. Every one of