2015_SummerScholarsProgramPresentation- Daniel Donchev
1. Daniel DonchevDaniel Donchev
Wilson High School, Long Beach; University of Southern California
Michael K Jones, Ph.D.Michael K Jones, Ph.D.
Gastric Pathophysiology Molecular and Cellular
2015 Summer Scholars Research Presentation
2. Survivin
• Survivin is a member of the
inhibitor of apoptosis (IAP)
family. The survivin protein
functions to inhibit caspase
activation, thereby leading to
negative regulation of
apoptosis or programmed cell
death.
• In the event of a stomach
ulcer, survivin is up regulated
to assist with the angiogenesis
process.
• Survivin inhibits caspase 3,
helping cells move indirectly.
• Our goal is to prove survivin’s
critical role in the angiogenesis
process and cell migration.
3. Objective and Hypothesis
• Objective:Objective: To elucidate the cellular and molecular
mechanisms of gastric (stomach) wound/injury (e.g.
ulcer) healing.
• Hypothesis:Hypothesis: Survivin as an inhibitor of caspaces
during apoptosis and cell division also has a tertiary
function; it can be involved, indirectly, in
angiogenesis and cell migration, and is crucial in
wound closing.
4. Means and Methods
• Means:Means: Create gastric-specific and vascular-specific
genome editing and “gene rescue” lentiviral transfer
vectors; and, to express cDNA corresponding to genes
hypothesized to be downstream effectors of survivin.
• Methods:Methods:
- Conduct BLAST search to identify promoters of interest
in the rat genome homologous to known human
promoters.
- PCR clone the promoters for: 1) VE-Cadherin (vascular-
specific), 2) TFF2 (gastric-specific), 3) EGFR (gastric
ulcer-inducible); and, 4) Flt1 (angiogenesis-inducible) and
introduce PacI and XbaI (SpeI) restriction sites.
5. Polymerase Chain Reaction (PCR)
• Relies on three principals of molecular biology:
1.1. DenaturingDenaturing - melting double stranded DNA template
into single strands.
2.2. AnnealingAnnealing - complementary DNA strand hybridization
via DNA primers.
3.3. ExtensionExtension - DNA strand synthesis via DNA polymerase.
6. PCR Continued
• The enzyme used in PCR is DNA
polymerase.
• A special reaction buffer is also required,
called a master mix.
• Two template strands are created from the
original template after each complete cycle
of the strand synthesis reaction.
10. Results and Our Next Steps
• Results: The promoters for VE-Cadherin
VE-Cadherin and EGFR were successfully
cloned into the TA cloning vector.
• Next Step: Isolate the cloned promoters
by digest/gel extraction and ligate them
into the CRISPR/Cas9 and FUGW
vectors.
11. VALBHS Research Translates
From Drug Discovery To Patient Care
• In this program, students learned methods
that are commonly used in labs to analyze
and research many different diseases.
• The research done in the lab translates
into discovery of new treatment plans, or
even new drugs that can be used to treat
patients.
Editor's Notes
Survivin is necessary for cell division and is an anti-apoptosis protein. It’s also expressed during embryonic development and is thought not to be crucial during the adult life.
We believe that it is expressed in some tissues during adult development including the stomach. In the event of a stomach ulcer, the surrounding tissue upregulates survivin, which plays a crucial role in the healing process.
Angiogenesis is the growth of new capillary blood vessels in the body and is an important natural process used for healing and reproduction.
Our hypothesis is: Survivin as an inhibitor of caspaces during apoptosis and cell division also has a tertiary function; it can be involved, indirectly, in angiogenesis and cell migration, and is crucial in wound closing.
Survivin helps remodeling tissue grow and divide. Survivin is upregulated in cells that are migrating. Some factors are targets of caspace destruction. If survivin is upregulated, it inhibits caspaces so they aren’t destroyed. IAPs (e.g. survivin) bind to effector caspases and inhibit them from cleaving their targets (but IAPs do not “destroy” caspases). If caspases are “over inhibited,” this creates a state conducive to tumorigenesis and cancer. If caspases are “under inhibited,” apoptosis would be abnormally high and could possibly impair the processes of wound/injury healing.
Means: Create gastric-specific and vascular-specific genome editing and “gene rescue” lentiviral transfer vectors to knockout genes hypothesized to be involved in ulcer healing (i.e. Survivin) and, to express cDNA corresponding to genes hypothesized to be downstream effectors of survivin (e.g. integrin α6/β4, focal adhesion kinase) as well as mutant forms of survivin.
Methods: Conduct BLAST search to identify promoters of interest in the rat genome homologous to known human promoters. . Rat promoters were chosen because the in vivo model of study is the rat. PCR clone the promoters for: 1) VE-Cadherin (vascular-specific), 2) TFF2 (gastric-specific), 3) EGFR (gastric ulcer-inducible); and, 4) Flt1 (angiogenesis-inducible) and introduce PacI and XbaI (SpeI) restriction sites. Use these constructs to exchange (by directed restriction site ligation) the ubiquitous/constitutive hUbC promoter in the CRISPR/Cas9 genome editing vector, pLV-hUBC-Cas9-T2A-GFP; and, the lentiviral expression vector, FUGW.
Denaturing – Before DNA synthesis can begin the double stranded DNA template must be unwound and separated into single strands. In cells, this is carried out by a family of enzymes. In PCR, heat is used to melt apart, or denature, the double stranded DNA template.
Annealing – Before a target region of DNA can be amplified, you must determine short sequences of DNA upstream (at the 5’ end) and downstream (at the 3’ end) of the target loci region of interest. These areas are then used to make short pieces of DNA called primers, which are complementary to regions upstream and downstream of the target loci region. Primers serve as start and stop points for amplifying the target loci region of the DNA to be copied. The annealing temperature is very important and we were able to find the temperatures we needed by going onto the company’s website and locating it there.
Extension – Primers are needed because the DNA polymerase requires an already existing nucleotide chain to bind and add nucleotides to one at a time. Once the polymerase locates and binds to template DNA and the primer, it initiates the addition of nucleotides and synthesizes new copies of the double stranded DNA template by adding nucleotides onto the primer and extending it. Therefore, primers provide a starting point for the DNA polymerase.
DNA Polymerase must be thermally stable because PCR cycles vary between temperatures of 52 degrees Celsius and 94 degrees Celsius.
The master mix contains all of the components for PCR to occur, including the individual building blocks of DNA, a special buffer to maintain optimum pH, salts, and Magnesium ions. It’s important to keep the master mix cold before use, so that the enzyme doesn’t start to work before you add your DNA templates.
It is called the Polymerase Chain Reaction because exponential growth of the number of template molecules occurs after each cycle is complete. After 35 cycles, the DNA of interest has been amplified sufficiently to be visualized using gel electrophoresis. This allows us to determine the presence or absence of the desired PCR products.
A method that separates large molecules (including nucleic acids or proteins) on the basis of size, electric charge, and other physical properties. The large molecule we separated was DNA.
To prepare DNA for gel electrophoresis, the DNA is cut into smaller pieces when mixed with restriction enzymes.
This process is called restriction digestion, and it produces a range of DNA fragments of different lengths.
During electrophoresis, molecules are forced to move through the pores of a gel, when the electrical current is applied.
The frictional force of the gel resists the flow of the molecules, separating them by size.
Steps in the process of gel electrophoresis are:
A tray is prepared to hold the gel matrix.
A gel comb is used to create holes in the gel. The gel comb is placed in the tray.
Agarose gel powder is mixed with buffer solution (10x TBE) that carries the DNA in a stable form. The solution is heated until dissolved and poured into the tray and allowed to cool.
The gel tray is placed in an electrophoresis chamber and the chamber is filled with buffer, covering the gel. This allows the electric current from electrodes at either end of the gel to flow through the gel.
DNA samples are mixed with a “loading dye” to make the DNA sample visible. The dye also contains glycerol or sucrose to make the DNA sample heavy so that it will sink to the bottom of the well.
A safety cover is placed over the gel, electrodes are attached to a power supply and turned on.
When the dye marker has moved through the gel, the current is turned off and the gel is removed from the tray.
The DNA molecules are made visible by staining the molecules with ethidium bromide which binds to DNA and will fluoresce in UV light.
VE-Cad, PacI-AgeI Promoter Minipreps 4: The left well next to the marker worked. TA vector is at the top, and the promoter is at the bottom. It’s cut with Bam and is diagnostic. They allow us to determine what clones that we used had the insert of interest.
Bottom left:
Bottom Right: Diagnostic, Mini preps using BAM. Testing for what has our insert. BAM digest.
Top right: PCR when I was getting the promoters. They have the right flanking restriction sites that we incorporated on the PCR. Bottom left and top right are the same bands.
Bottom left: We need to purify the DNA and purify the big backbone of the vectors, and we will ligate them together, directional ligation.
Left: Third well is the correct digest, Sixth well is also a correct digest. They have the correct size.
Right: Crispr cas 9 vector is well 1. Next 4 wells are the vectors that we will ligate in our RNA guides for the crispr cas 9. Will be used in conjunction with well 1 ( in our golden gate cloning. (removes the restriction sites, and leads to the products you want) Cas9 will be expressed with four separate guide RNAs in the end.
Six well: expression vector.
Next three wells: We got the plasmids from addgene and grew them up.
The promoters for VE-Cadherin and EGFR were successfully cloned into the Results:TA cloning vector such that the proper flanking restriction sites were verified by restriction mapping.
The next step will be to isolate the cloned promoters by digest/gel extraction and ligate them into the CRISPR/Cas9 and FUGW vectors.
Site-directed mutagenesis (SDM) is an in vitro procedure that uses custom designed oligonucleotide primers to confer a desired mutation in a double-stranded DNA plasmid. There are many reasons to make specific DNA alterations (insertions, deletions and substitutions), including:
To study changes in protein activity that occur as a result of the DNA manipulation.
To select or screen for mutations (at the DNA, RNA or protein level) that have a desired property
To introduce or remove restriction endonuclease sites or tags
Used to knock out a PacI site because the vector contains two PacI sites.
The other reason for it is on the integrin alpha 6 beta 4 we want to knock out the target sites of caspace 3 to make the integrin impervious ( no longer a target of caspace3, can’t be cleaved)
Use it to mutate survivin so you can dissect the different functions of survivin with regards to the process of ulcer healing.
Do more digests with Pact I and Age I and PacI XBA I and PacI SPE I, and isolate the promoters with a low melt gel.
We will do the ligation.
