Download SURF 2010 Prospectus.doc

Nicolas Aguilar
002:199, Spring 2010
University of Iowa Department of Biology
Dr. Marlan Hansen
UI Department of Otolaryngology
200 Hawkins Dr, 21163 PFP
Iowa City, IA, 52242-1078
Direct Evidence Of Neurofibromatosis II Protein – Merlin – Inhibiting Glial
Growth Through The ErbB2 Receptor
Abstract
Merlin (Moesin-Ezrin-Radixin-Like)
is a tumor suppressor protein
predominantly found in nervous tissue. It is
a cytoplasmic protein that links actin
filaments to the cytoskeleton and helps
regulate cell signaling.
It is unknown how exactly Merlin
suppresses tumor growth. Nonetheless,
mutated, inactive Merlin can commonly be
found in neoplasms, including vestibular
schwannomas (see Figure 1).
Studies suggest that Merlin
correlates with cell cycle entry via
interactions with certain transmembrane
receptors, like Human Epidermal Growth
Factor Receptor 2 (ErbB2).
To provide direct evidence of Merlin
interacting with ErbB2, a DNA construct
with Merlin tagged with CFP (cyan florescent
protein) will be biologically engineered. Cells
lacking functional Merlin will be transfected
with this construct. Then using fluorescent
resonance energy transfer (FRET) microscopy,
Merlin’s molecular interactions with ErbB2
will be monitored.
Figure 1. Human MRI of Type II Neurofibromatosis
patient with bilateral vestibular schwannomas
(University of Iowa Department of Otolaryngology)
Neurofibromatosis II Protein – Merlin – Inhibiting Glial Growth Through The ErbB2 Receptor 2
Materials and Methods
The intent of this research project is to provide direct evidence that Merlin and ErbB2
interact. The approach will be observing protein interaction via a CFP (cyan fluorescent
protein) tagged Merlin DNA construct. In brief this involves cutting out functional Merlin
and CFP then taking these genes and inserting them into an antibacterial vector to be
inserted and selected for through bacterial culturing. The functional and tagged Merlin will
then be inserted into human schwannoma cells lacking functional Merlin and using FRET
microscopy, interactions would then be observed.
Specifically, this approach requires the following steps.
Maxiprep. Bacteria containing the CFP (eCFP-N1) plasmid cultured overnight in growth
buffer (Dr. Steve Green, University of Iowa Biology Department). This is done to produce a
large number of the plasmid. To isolate the now abundant plasmid the protocol from the
GenElute HP plasmid maxiprep kit was used (Sigma Aldrich). Once isolated the DNA was
mixed with elution buffer at 40 degrees Celsius to facilitate re-suspension.
Nanodrop Spectrophotometer. To measure the amount and the purity of plasmid, a
nanodrop spectrophotometer will be used and the ng/ µL and 260/280 ratio recorded. DNA
absorbs UV light at 260 and 280 nanometers while aromatic proteins absorb UV light at
280 nm. This known ratio of 1.8 for DNA allows for the measure of the purity of DNA.
Experimentally detrimental protein contamination would correspond to a ratio of lower
than 1.8.
Restriction Enzyme Digest preformed using the purified CFP plasmid, wild type Merlin (wt),
Merlin (S->A), and Merlin (S->D) from University of Iowa Biochem Stores (Iowa City, IA).
Merlin wt is functional Merlin. Merlin S->A is mutated Merlin with a functionally important
amino acid Serine point mutated to Alanine. Merlin S->D has that Serine mutated to an
Aspartic Acid. A restriction digest uses enzymes that recognize specific sequences of
nucleotide base pairs and cuts DNA at these locations. These recognition sequences are
typically six, eight, ten, or twelve nucleotides long. The ultimate purpose of this digest is to
cut out the CFP and cut the three kinds of Merlin (wt, s->A, and S->D) so the CFP could
eventually be attached or ligated. Restriction enzymes Xho1 and EroR1 were chosen on this
basis (University of Iowa BioChem Stores, Iowa City, IA).
Gel Electrophoresis using 1% Agarose gel with 1% TAE buffer will be made. Cut DNA will
then be stained with 10x Blue Juice then pipetted into small wells on one end of the gel
slab. The gel is then subjected to an electric field (120 V). The negatively charged DNA is
drawn towards the positive spectrum of the electric field. Molecules travel at different
rates through the gel based on charge and how well fragments can maneuver through the
gel. Since none of the four nucleotide bases carry a charge, distance traveled by DNA
fragments is soley contingent on the size of the molecule. As a note, the negative net
charge of the DNA is due to the sugar-phosphate backbone thus gives all fragments an
equal negative charge. The size of the DNA pieces can then be determined by comparing
against a 1 Kbp DNA ladder. After running the gel, the DNA will be tagged with Ethidium
Bromide. When EtBr is bound to DNA it is 20x more florescent under UV light. This makes
taking pictures of the progress of the DNA possible. It is vital to check that the DNA does
Neurofibromatosis II Protein – Merlin – Inhibiting Glial Growth Through The ErbB2 Receptor 3
make move across the gel because this confirms that the chosen restriction enzymes and
protocol are working correctly.
Ethanol Precipitate. Desired DNA bands can then be identified and cut from out of the gel
using razor blades. DNA is then separated from gel and purified through an EtOH
precipitate protocol using NaCl and EtOH. Again the Nanodrop Spectrophotometer should
be used to check ng/ µL and 260/280 ratios are check.
Ligation. First a ligation calculation must be preformed using several variables and an
online ligation calculator (for example http://www.insilico.uniduesseldorf.de/Lig_Input.html). Numbers that should be used are as follows. Vector base
pair number: 4700. This is the size of the CFP vector. Insert bp number: 1800. This is the
size of the Merlin insert. From the nanodrop machine, the ng/ µL for each of the three
Merlin types must also be entered. Finally, since this is a cohesive end ligation a ratio of 1
mole of vector to 3 moles of insert will be used. Vector and DNA are then combined with a
10x buffer, a T4 ligase, and distilled and deionized water, and then incubated for 5 minutes
to coax DNA ligation.
Transformation of DH5 α Cells. Ligated DNA (wt, S->A, and S->D) is added to bacteria of
DH5 α cells through a heat shock process. The bacteria are raised from 0 to 37 degrees
Celsius as a heat shock for 45 seconds to open the cell membrane and allow the ligated
DNA to enter. Bacteria are then cultured which allow the DNA plasmid CFP vector to be
replicated by using the cells machinery.
Cell Culture. The DH5 α bacteria cells with CFP/Merlin plasmid (which also has pre inserted
antibiotic resistance) are plated on LB Agar petri dish plates with the antibiotic
Kandamycin. Cells that survive would be confirmed to have taken up and replicated the
vector. Next is to purify the plasmid and insert it into another vector K-NpA. This would
then be sent to ViraQuest Inc in North Liberty, IA, to create an adenoviral vector. This virus
would then to be used to infect human cancer cells (schwannomas with a nonfunctional
Merlin protein) with a CFP tagged Merlin to observe whether or not it interacts with ErbB2.