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Project Proposal
Design and Construction of a Transgenic Animal Model for Non-Invasive Measurement
of Angiogenesis
Advisor: Dr. Duco Jansen
December 2, 1999
Katie Krochak
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Project Proposal
Fries Format
Project Title: Design and construction of a transgenic animal model for non-invasive
measurement of angiogenesis
I. Project Overview, Objectives, Major Milestones, and Schedule
The major objective for this project is to optimize laser parameters in order to
minimize tissue damage in medical procedures and such. This will be done by designing
and constructing a cell line with several genes. VEGF is the promoter used in this case to
control the other two genes to be inserted. Gfp, or green fluorescent protein, is a signal
protein which will luminesce when activated. Luciferase is the another gene which
causes the cells to produce light when activated. The difference between these two cells
is the kind of light which they produce.
The major milestones in this project are as follows:
Maintaining the 3T3 cell line
Creating the construct
Transfecting the cells with the construct
Cloning the transfected cells and keeping them alive
Characterizing the cells for kinetics of response
This project overall should take 9 weeks. My activities in this project are planned
to start at the beginning of January when classes resume. This is assuming that the
construct of the three genes to be placed in the DNA has been made already. The cells
throughout this process will be stored in an incubator in Dr. Haselton’s laboratory and
will be split as needed, usually around twice a week in order to maintain appropriate
levels of the cells for the environment.
The first week the 3T3 cells will be thawed and maintained in media in the
incubators.
The second week the thawed cells will be co-transfected with the construct of the
three genes, VEGF, Gfp, and Luc and the antibiotic, geneticin. They will be nourished in
the media and stored in the incubator.
The third week the cells will be nourished with media and split if needed. The
cells during this week will also be selected for the geneticin resistant gene on the
plasmids using geneticin. This ensures that only the cells with the construct inserted,
which are resistant to the antibiotic, grow.
The fourth week the selected cells are maintained in the incubators in fresh media.
During the fifth, sixth, and seventh weeks, the only thing that occurs is the
picking of colonies of the cells (hopefully with the genes inserted). The cells are then
grown in flasks of increasing sizes as the rate of growth increases and as the abundance
of cells requires.
During the eighth week the new cell colonies will be tested for containing the
promoter VEGF. It is assumed that if the test is positive for the promoter, that the other
two genes have been inserted and accepted by the cells as well. This is done by exposing
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the cells to stressful conditions such as hypoxia. This will induce tissue damage and will
activate the promoter.
The ninth week the cells are characterized to determine the kinetics of response.
This is done by once again subjecting the cells to hypoxic conditions hoping to induce the
promoter. This is important in that is helps to optimize the experimental conditions in
order to make the actual experiments easier to complete.
After this point, these cells are considered to be a stable cell line and can be used
in laser experiments as other cell lines are.
II. Market Need and Market Potential
This project must be accomplished in order for Food and Drug Administration,
ISO 9000, and EC to approve new laser procedures. This will broaden the range of laser
uses. In addition, these experiments will determine more about the behavior of cells once
lasers have touched them. This in turn should help to identify the reasons for and to
alleviate some of the side affects, which exist as a result of laser procedures.
Laser procedures have become a huge industry already and this area has only just
been founded. In medicine, physicians can use lasers to make incisions, vaporize tumors,
close blood vessels and lighten the skin around birthmarks and such, selectively reduce
pigmentation, treat skin wrinkles, or even for hair transplants. (reference) This field not
only encompasses these cosmetic procedures but also dentistry and ophthalmology.
In dentistry, lasers are used in order to help whiten teeth. Instead of home
bleaching which takes from 3 to 4 weeks of spending several hours in plates of gels or by
using different tooth pastes, these procedures take around 2 hours and can be done in
office. They also allow for more individualized attention to teeth which are worse than
others while the at home kits treat all teeth regardless of their condition.
In ophthalmology, lasers are used in several procedures. Not only does the use of
lasers make the procedures more accurate and dependable, but they also make the
procedures easier on the body by decreasing the amount of bleeding and the stress placed
on the body during surgery (i.e. through anesthesia and other processes). Procedures such
as LASIK, PRK, RK, AK, and ALK all involve reshaping the cornea in order to improve
the patient’s vision. These are just a few of the procedures that involve lasers. There are
many more.
Because this field is so large and is ever becoming larger, this research into
improving the techniques and conditions lasers can be used under is of huge value to the
medical field.
Unfortunately, all of these results are indirectly related to this project. The
development of this cell line will not specifically lead to any huge profits or major
developments in the laser industry. Instead it will allow for people to study the effects of
lasers on the cells. These cells will not be sold, but if desired, given to the researchers.
The mice once transfected, will not be sold either, but given away as well.
III. Product Proposal
This project involves first the development of a stable cell line with the desired
genes, VEGF a promoter, Green Flourescent Protein, and Luciferase, inserted. From this
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point, many experiments can be done on these cells in order to determine how the cells
react and ways in which to better use lasers in industry. After cell development, these
cells can be inserted into mice and from here experiments can be preformed on the mice
in order to prove that the procedures and such desired as a result of the cell experiments
will work on animals.
IV. Strategic Fit
This cell line fits into the experiments by creating a different environment for the
experiments to take place under. One can discover much when using a single set of
conditions, but changing one factor could change everything. In order to increase the
opportunities, under which these laser procedures can and should be used, different
factors need to be investigated.
V. Risk Analysis and Research Plan
This specific project involves few risks. Technically, this project has little risk. It
is a difficult, unpredictable procedure and never can it be stated for sure that a cell will
accept, grow, and reproduce with the new genes in it. Because this is not a product, which
will be competing with others in the market place, there are no market risks. There are no
considerable financial risks involved either at this point. Those come into the picture
when the procedures are to be developed and put before the FDA and other councils for
approval.
VI. Economic Analysis
This specific project of developing the cell line and then the mouse line is not
very pricey. It does involve the use of some expensive equipment, but this equipment is
common and found in most laboratories. For example, mocropipettes are needed to make
the transfections, gel holders to run the gels in order to determine nucleic acid and protein
separation, and UV light boxes, to view the stained nucleic acids, are all needed. In
addition, the proteins to be fused into the cells are needed as well as the medium to grow
the cells in, the vectors to insert the construct with, and other miscellaneous materials
such as the flasks, agarose and EtBr to run the gels with. Overall, the total cost of this
project is $5000.
VII. Core Project Team
Working on this project will be myself, Katie Krochak and a graduate student
from Dr. Tom Daniel’s laboratory in the Vascular Biology Center. The graduate student
has strengths in creating the construct and a more thorough knowledge of the
mechanisms which should be followed and the conditions under which success is
expected. I am aware of the procedures that must be followed in order to maintain the cell
line and in testing for the appropriate cells once some have developed. Finally,
overseeing this project is another graduate student, Jen Baran, who has completed this
procedure before and can guide us towards its completion, and Dr. Duco Jansen.
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Design and construction of a transgenic animal model for non-invasive measurement of
Angiogenesis
Description: In collaboration with the Vascular Biology Center (Dr. Tom Daniel) a
student will work on the design of a genetic construct carrying the promotor for VEGF
(vascular endothelial growth factor) and firefly luciferase as a reporter gene. The
ultimate goal of this project is to establish an animal model based on transgenic
techniques in which angiogenesis (the growth of new blood vessels) can monitored by
detecting light produced by luciferase using our recently acquired photon counting
imaging system (luciferase is an enzyme that is found in fireflies and causes them to
glow). The application of this animal model will be in laser-irradiated wounds and tumor
growth. This student will work directly with a post-doctorate student in Dr. Daniel's lab
and a graduate student in my lab.
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