Matt Boyle, Entry #5, Horse Round 2 and Last Day

After some thorough research, I found that equine IL-4 is nearly 5 times less effective of a protein than canine IL-4.  In the first round of experiments, I had been putting an amount of IL-4 into the wells equivalent to that of the dog experiment (2ml IL4/ml of media). However, if the equine protein is less effective, a much higher concentration of IL-4 must be added to the cells in order to induce a similar response.  We also decided on a new formula for growth media, consisting of horse serum as opposed to the original fetal calf serum, and pen strep as an antibiotic as opposed to what we used the first time (gentamycin), for there are published papers about people culturing equine B Cells using pen strep.

We are also adding a component to this experiment.  Some of the B Cells are going to be CFSE labeled.  This will tell us if cells are not proliferating at all, or if they divide and then die off immediately.  CFSE dies the cells a dark green and with each division of the cells the intensity of the green is cut in half.  Therefore we can flow the cells and the machine will tell us how intense the green is in relation to that very dark shade.  This will tell us if we are not getting any proliferation at all, or conversely, is the cells proliferate but their living conditions are such that the cells cannot survive.

New blood arrived on the Monday of my last week and again the PBMCs were again isolated and plated with and without feeder cells expressing CD40L.  They have been cultured with the new growth media and received a more concentrated dose of IL-4 so we will see how this one goes!

The Mason lab is embarking on a new project. Led by the PhD student Kazim, the first infusion of CAR-T Cells will be infused into a German S

heppard with lymphoma named Lady.  The CAR T Cell project is really remarkable as it uses the HIV Virus to infect healthy T-Cells that become “serial killer cells” that seek out and kill tumor cells.  Carl June, a revolutionary researcher at Penn, has done some incredible work with CAR T cells and you should really watch this video if you have 5 minutes. Its so cool.

https://www.youtube.com/watch?v=h6SzI2ZfPd4

Ms. Cozine came to visit me last week which I thought was pretty funny because the last time she saw me in a science setting was when I was in her freshmen chem honors class! I taught her how to count cells via hemacytometry, we made LB Agar plates, and even made some more 70% EtOH (ethanol) solution before meeting everyone for lunch.

 

Hemacytometry -- a 10ul sample of the cells in their growth
media is put in this little glass frame containing a tiny tiny grid that looks
like this under the microscope--you count all the cells within the boxes and
plug that (when multiplied by the volume of the media in the vial)
number into a big equation that gives you the total cell count in
the media.

A picture of my building I snagged on my way home on my second to last day.

Wednesday, my last day, was incredibly sad.  We all went out to dinner at Mad Mex and my friends from the lab gave me presents, including a necklace made from the exact vial from the liquid nitrogen freezer that once contained a type of cell that I mistakenly thawed and cultured.  Pretty funny.  Everyone has tried to convince me to go to Veterinary School while I have been here and they even gave me a Penn Vet T-shirt.  EXP has easily been the coolest experience I have had in any summer and I could not have asked for better people to work with, a cooler topic to study, or cooler city to do EXP in! From Phillies games to Cheesesteaks to the Palestra, and meeting my fellow EXPers in the city, it was all pretty cool.See you all in September!

Met Bridgid for a Phillies game.

Matt Boyle, Entry #4, Do We Only Cure Dogs? "Nayyyyy."

After all this talk about CD40 and K562s that are irradiated and culturing the cells, I realized I have never explained to you guys what CD-40 B Cell activation is.  My bad.   So here we go.

Cd40 B Cell activation is a method by which B Cells become antigen presenting cells.  The binding of CD40 to CD40 Ligand activates the cells, thereby increasing the ability of the cells to present foreign antigen to the immune system.  In the, the PBMCs of an organism are extracted and cultured with these feeder cells (from the K562 cell line) expressing CD40L.  In the presence of the cytokine interleukin 4 (IL-4) the activated cells proliferate, allowing for a large number of cells to be grown from a small number extracted from a blood sample.  This is extremely advantageous because, as each vaccine is specific to the host the blood was taken from, just a small amount of B cells from a small blood sample can grow into a huge amount of antigen presenting B cells that will increase the immune-response to the cancer.

The problem is (and the efforts of my project are based around) the feeder cells we have in the lab express HUMAN CD40L.  Canine CD40 recognized the human CD40L but we do not know if the horse CD40 will.  I performed a blast search on the homology of canine and equine CD40 and the proteins are approximately 70% similar so nothing is guaranteed.  Should the CD40 of the equine cells bind with the human CD40L, the cells will be activated and their proliferation will be sustained in the lab until B-Cell count reaches a threshold of viable cells able to be infused back into the horse.  Then the cells will be electroporated with the RNA from a tumor sample of the horse removed via a biopsy.  The B Cells will then present the tumor RNA as an foreign antigen to the immune system, and upon re-infusion into the horse (the same horse that the B Cells came from) its immune system will recognize the tumor as foreign, thereby educating it to recognize and kill the rest of the tumor cells.

An animation of the CD40 B Cell Activation. The little orange things on the sides is RNA being electroportated into the cells before re infusion of the B Cells into the patient 

The science behind the protocol is truly fascinating but that is only half of the reason I am enjoying EXP so much. The other half is because of the people I get to work with.  Dr. Mason Is truly hilarious; for example, when the horse blood came in she was walking around saying “NAYYY.”  Dr. Mason also just got a full-time vet to help her with the trials in the clinic (her name is Martha and she just graduated from Cornell’s Vet School).  We all went out to dinner in Philly for Martha’s first day where Dr. Mason was telling us about how she was invited to Richard Lichter’s dog’s birthday party on Saturday.  Richard Lichter is a multi-millionaire who owns Mel Gibson’s old estate in Greenwich Connecticut with 16.5 bathrooms) and his goal in life is to cure equine lymphoma.  He is an enormous benefactor for Penn’s veterinary research and has a party date set (April 16) for the celebration of Dr. Mason having cured canine lymphoma.  Upon hearing this, Dr. Mason decided we must get to work as cancer is certainly not cured yet. We sat outside at dinner and Dr. Mason was directing a girl to parallel park next to our table when a group of people came out of the restaurant in a huge fight.  People were screaming at each other and Dr. Mason did not miss a beat as she stood there like one of those people on the runways at airports helping this girl parallel park.  It was probably the funniest moment from my time in the lab.

As for the horse cells, they look horrendous. But not really.   If you look through the microscope at the K562 Wells in relation to the KtCD40L Wells, there is significant more clumping in the CD40L wells, leading one to believe the B Cells were indeed interacting with the human CD40 protein.  However, upon analysis, the B Cell populations were almost completely dead (as you can see in the flow graph).  Despite this failure, the way the cells looked through the microscope was very promising.  The cells seem to be interacting with the protein but not proliferating, which could be very closely related to an insufficient amount of the cytokine IL-4 that causes proliferation or perhaps there is insufficient serum, insulin, or other component of the growth media.

The K562 (left) and KtCD40L (right) wells as they appeared through the microscope.  There is obvious clumping going on in the KtCD40L wells; significantly more than in the K562 wells, leading one to believe that the equine CD40 is indeed interacting with the huamn CD40L.

 These the flowcytometry graphs pertaining to B Cell population in one horse (R600). You can see from the day the bloods arrived (left) that the B Cells made up 17% of the living PBMCs, but on day 7 when the cells were flowed again, the percentage of B Cells had shrunk to only 3.12% of the population.  This is an opposite result to what we thought was the case by looking at the cells through the microscope.

The plan from here is to get new horse blood and determine which components we will change from the media or in what is added to the wells to initiate B-Cell proliferation.  Now I am staying for an extra week to have some time to see round two through to completion.

Matt Boyle, Entry #3, Working Like a Dog (or should I say horse)

The plan was to get samples of horse blood last Wednesday until Dr. Mason realized that we did not have enough irradiated k562 cells (irradiated means the membranes are sort of stuck in time but the cells are still alive) for the control of the experiment.  The problem was that irradiated K562 cells expressing CD40 Ligand had been in the liquid nitrogen for several months and when we irradiated the K562 cells we wanted to put them in the liquid nitrogen as well to make the conditions as similar as possible to the CD40L expressing cells; who knows the effects that freezing a group of cells down to          -150⁰ dos  to the viability of cells.  Therefore, we postponed the blood delivery to next Monday I had to go into the liquid nitrogen and thaw out a vial of K562’s, grow them to a total of three million, irradiate them, and freeze them down.  By the weekend, they were in the liquid nitrogen and had been in identical conditions to the CD40L expressing cells.

Speaking of the irradiator, going to use that machine is an intense process.  The irradiator is located in the basement of my building which also happens to be the rat facility (that you can smell every day from the stairwells).  Once you get downstairs you need to put on little boot things and a hairnet and gloves and then at another station you must put on a sterile gown that looks like you are scrubbing in to perform a surgery.  Then you must stand in front of a machine that does a retina scan that allows you to open the door with a separate key.  I told you it is intense.   Dr. Mason took me down once and I wasn’t even allowed in the room.  She scanned her retina, entered the room, and I had to watch from outside.

Dr Mason at the irradiator, in her sterile gown and hair net, as I look on through the small window in the door.

 On Monday morning, the blood of three horses was pulled at New Bolton Center and shipped to Dr. Mason’s lab.  Dr. Mason made sure it would be “hand delivered” as to ensure optimal condition of the blood.  The members of the lab that are participating in the horse lab (myself, Dr. Mason, lab tech Josephine, and PhD student Kazim) had a quick meeting to make sure everyone knew what their role was and how the bloods would be handled.

The three horses all had labels to identify them by (R600, R665, and R289) however their given names were also written on the tubes of blood – Wise guy, Class Fantacy (spelled like that), and Idol – which I thought was hilarious.

The vials in which the horse blood arived.  You can see that each horse had an ID number as well as a given name that was usually pretty funny.

I was in charge of extracting the PBMCs by using a protocol that I had discovered was successfully used in a lab in 1978 to extract equine PBMCs over ficoll.  We did not know if this 1978 procedure would be more effective than the protocol the lab uses for the dogs.  We had 20ml of blood from each horse so we used a single sample of 10ml to decide which protocol we would use.  We got decent yield of PBMCs from the 1978 protocol which was around three million.  We attempted PBMC isolation with another 10ml of blood, this time with Dr. Mason’s protocol, and got a yield of approx. 8 million cells and therefore used her protocol on the remaining 20 ml. 

The equine PBMCs are pretty visible -- they are the clear carpet-like layer right below the yellow (which is serum)

The PBMCs were then cultured under three conditions.  For each horse, a given number of their cells would be left alone to see if the B Cells proliferate on their own (obviously they will not, this is just a formality to say we evaluated this).  The next set of wells contains The PBMCs cultured with K562 feeder cells.  This is the control for the following row in which the PBMCs are cultured with K562 cells that express CD40 Ligand.  The only difference between the last two rows is the expression (or lack thereof) of CD40 Ligand on the surface of the feeder cells.  Therefore, if there is a difference in growth of B cells among these rows, it HAS to b

This is a diagram of the plate that the cells were cultured in.  For the three different horses, there were three different conditions that the PBMCs were cultured in (by themselves, with plain feeder cells, with feeder cells that express human CD4

We will check the cells on days 2,4, and 7 and add new growth media and cytokines so by next week

we will be able to stain flow them (mark with antibodies and computer analyze) to determine the content of all the wells.  

Julius Sim, #5, Summary about Last Week at Penn

I finished my last week at my lab at UPenn under the supervision of Dr. Carlo Ballatore, working on microtubule-stabilizing agents as Alzheimer's disease therapeutics.

Azza- Entry #5

I have learned an incredible amount while I was at the Lab. It was definitely a great experience! I had the opportunity to transform, transfect and ligate bacteria. Grow small scale and large scale bacterial cultures. Work with mice and observe the work other lab members spent with zebrafish. I performed many minipreps and midipreps. I became super comfortable with PCR and gel electrophoresis. I also got to learn how to make and use protein gels and interpret the results .I also got to grow crystals, which an essential step in understanding the biophysics and biochemistry of the mutant protein or wild-type portein that you are researching.  I walked into UCSF knowing the basics such as PCR and gel and I left with a much greater understanding and appreciation for lab work. My graduate student taught me so much. I got to work with him while we focused on the main project of the lab and he also assigned me solo projects to work on. It was a fantastic experience

, and I would love to do it again!!!

Azza- Entry #4

One of my side projects was getting to make more of an enzyme called phusion that is used in PCR. Phusion is similar to the Taq polymerase were more familiar with, but the difference is that phusion is a lot more heat stable because it was first discovered in hot springs and therefore can withstand high annealing temperatures. It was definitely not easy trying to purify phusion with the FPLC, but once I was finally able to the amount of phusion I was able to replicate was worth up to $168,000! Taq is available for commercial purchase, but it’s also very expensive, so most labs (if they have the time) prefer to make more phusion on their own. The process is very straight-forward. You began with a phusion stock that is stored in the -80 degree. You use a pipette tip and scrap some phusion out and place it in a 25mL starter culture of broth (either LB or 2XYT). Because phusion has a kanamycin and chloramphenicol resistance gene, we used 25 uL of each to have a selection component. The next day we use the starter culture to induct a larger culture of 750 mL of bacterial broth. Then we spin down the larger culture and suspend the pellet in lysis buffer and sonicate and heat it up and finally run it on the FPLC. The FPLC is a machine basically composed of anion and cation exchange tubes that allow for the purification of phusion. To test if we’ve purified phusion we run the sample on a protein gel. The image below are my results after performing the protein gels. The different wells are different concentrations of phusion which were diluted with nuclease-free water. The last well is the commercial phusion we used to compare the one I made to. You can see how concentrated the phusion I made, which shows that they can now start using the phusion I made! 

 

Azza- Entry #3

Since I've come into the Lab, I was given my own personal project to work on. The goal of this project is to clone a plasmid responsible for creating Cas9. Cas9 is a protein that is a part of the CRISPR/Cas9 method of cleaving out around 20b.p of DNA and inserting foreign DNA into a cell line or an organism in order to observe a mutation of a targeted gene. Cas9 is an RNA guided DNA endonuclease enzyme. To clone CAs9 we are using a plasmid created by Martin Jinek, who once worked for my PI here at UCSF. Martin Jinek was also the student working for Jennifer Doudna when she discovered CRISPR/Cas9. Jinek is also the first author on some very famous papers that introduced the world to CRISPR/Cas9. The way I started off with this project was to convert the word document I've received from Martin Jinek for the coding of the MJ922 plasmind to an application called ape which lets you annotate certain regions of the plasmid. Then we got the plasmid from a postdoc student in the lab and transformed it using Rosetta cells. Rosetta cells are probably the most versatile cells used for transfection and transformation. Once we transformed the cells, we grew them at large scale. Then we spun them down and created large cultures and then extracted the  protein from the pellet using the FPLC machine. I only got so far with this project as time was running out and I wanted to stick with a different project of cloning and purifying more Phusion. 

this is what the annotation of the plasmid looked it, just to familiarize myself with what was being transformed.