Facioscapulohumeral Dystrophy SEP Project How restriction enzymes and gel electrophoresis can play a role in treating a disease.

Review yesterday What was the charge of the food coloring molecules? Which moved the farthest? Why? How does yesterday’s lab compare with running restriction fragments in a gel? Any questions about learning objectives #23 – 26 or your notes about gel electrohporesis? From this point on, I’ll assume you know all this information. Notes on today’s PowerPoint are required – paraphrase!

Fred Hutchinson Cancer Research Center - Intro Spent several weeks in a lab there in summer 2013 Will use experience to show how manipulating DNA with electrophoresis (and more) can help solve human problems – Focused on a type of muscular dystrophy LO #29: Be able to determine if a DNA molecule contains a gene by digesting and electrophoresing it. LO #30: Explain how inserting genes into model organisms can help us determine how proteins function.

Facioscapulohumeral Dystrophy (FSHD) Background – FSHD patients - deteriorating muscles in upper body – 3 rd most common form of muscular dystrophy – Autosomal dominant (one copy of a bad gene = disease)

Facioscapulohumeral Dystrophy (FSHD) Background – Everyone has the Dux4 gene in all of their cells – FSHD patients: “Dux4” gene is turned on Muscle cells in patients contain “Dux4” protein (bad) – Healthy humans: “Dux4” gene is turned off Muscle cells do NOT have the Dux4 protein (good) Turned on during early development, then turned off? Muscle cells in FSHD patientsMuscle cells in healthy individuals Dux 4 Protein No Dux 4 Protein

Research at Fred Hutchison Cancer Research Center Scientists’ “big picture” question: Can we cure or treat FSHD patients by finding a drug that either: 1) Turns off the Dux4 gene or 2) Inhibits the Dux4 protein? Use gel electrophoresis and restriction enzymes Use E. coli cells to help manipulate DNA So, how does all this work? – How can E.coli cells in a lab help find a cure?

In some FSHD patients: Test a potential new drug/medicine on patients Lab: Test potential drugs/medicines on human muscle cells with Dux4 protein “Big picture” question: Can we cure or treat all FSHD with a new drug? Lab: Insert “controllable” Dux4 gene into E.coli cells (very easy to use in lab) Lab: Insert “controllable” Dux4 gene into human cancer cells (moderately easy to use, immortal culture) Lab: Insert “controllable” Dux4 gene into healthy human muscle cells (complicated cells, but realistic) Success? Did we insert the gene? Success? Can we get the gene to make Dux4 protein? Success? Can we still get the gene to make Dux4 protein? We will measure success here! Success? Did any drugs work? Success? Did the drug work in some patients?

Upcoming lab Obtain “model” samples of Fred Hutch E.coli cells that have had controllable Dux4 gene (hopefully) inserted into them. Bacterial DNA samples called “plasmids” Use restriction enzymes to cut DNA Look for Dux4 gene in restriction fragments Plasmid (entire circle) Dux 4 gene Unrelated gene

Dux4 gene has a HindIII restriction site at either end Cut DNA samples with HindIII Use gel electrophoresis to separate fragments – Determine the size of these fragments E.coli cell WITH Dux4 gene E.coli cell WITHOUT Dux4 gene

Do any fragments correspond to Dux4 gene? If so, success! E. coli cells contain the correct gene! E. coli cells now can be used to further their research E.coli cell WITH Dux4 gene E.coli cell WITHOUT Dux4 gene Gel Electrophoresis

In some FSHD patients: Test a potential new drug/medicine on patients Lab: Test potential drugs/medicines on human muscle cells with Dux4 protein Can we cure or treat FSHD with a new drug/medicine? Lab: Insert “controllable” Dux4 gene into E.coli cells (very easy to use in lab) Lab: Insert “controllable” Dux4 gene into human cancer cells (moderately easy to use, immortal cultures) Lab: Insert “controllable” Dux4 gene into healthy human muscle cells (complicated cells, but realistic) Success? Did we insert the gene? Success? Can we get the gene to make Dux4 protein? Success? Can we create some Dux4 protein in experiments? We will measure success here! Success? Did a drug stop the protein? Success? Do patients improve? Can we prevent FSHD?

Questions? This PowerPoint is available in the documents section of my website. – “FSHD PowerPoint”