Week 2 Attachment Sharing

Hey there, i miss everyone because I'm alone here..haha, here's my part, if you don't understand, just ask me, if I never reply means I don't know what's the answer. lol

About my attachment

I’m attached to a research institute, in which all levels have research labs and different focus areas. I will be working on a paticular level throughout my attachment, and this level focuses on delivery of drugs, genes and proteins. Each student in my lab who are mainly university students and they are attached to a mentor, including myself and we are assigned onto a specific delivery. The major problem in current researches done throughout the world on such deliveries is that, the delivery efficiencies are low. Thus, we are trying to enhance the delivery of drugs and genes into cells for future cancer therapy and treatment of infectious diseases. Majority of the publications done by IBN are on cancer therapy.

Reseach Focus

My delivery focus is genes. These genes can be plasmid-encoding ones or siRNA. I’m working on how efficient the tranfected cells express the genes delivered to them and also the percentage of viability. Finally a comparison is done using different cell lines and nanocarriers. Let me explain the principle first.

Principle of the Project

This lab synthesizes nano-sized cationic polymers as carriers for the genes. The polymers have nitrogen group( NH₃⁺) which are positively charged that bind to the negatively charged phosphate group(PO₄^-) in the DNA. They form a transfection complex which will then be used to tranfect the cells. The uptake occurs via endocytosis. I will prepare the polymers and DNA solutions based on very confusing calculations in which I took almost a week to understand. The tranfection complexes are prepared in different conditions and pH(5&7). Conditions basically mean different nitrogen to phosphate ratio(N/P ratio: 0-40). I usually do 9-10 conditions. These are mainly done to determine the optimal concentration of the transfection complex and at what conditions do cytotoxicity occurs.

Lab techniques

Since I’m dealing with cells, I need to master all the cell culturing techniques and maintain my own cells. There are quite a no. of machine that I must learn to operate. Apart from that, I will have to prepare all my reagents by myself (labeled with my name) throughout my experiments and strictly no sharing.

Culturing and seeding of cells

After preparing all reagents and subculturing, I will seed the cells on 24 well plates or 96-well plates depending on which cell lines. Some cell line that I handle are HepG2, HEK 293, heLa and MCF-7 & etc. On the next day, I will do gene transfection on 1 cell line. The cell must meet the desired density, thus cell counting must be done using haemocytometer. Each condition has 4-6 replicates, thus every row of plate is for 1 condition. Then I will repeat the whole thing for another pH. The cells are then incubated in the CO₂ incubator for 4 hrs and then the medium are changed to stop transfection. I will then need to wait for 3 days to do analysis using MTT assay and luciferase analysis assay.

Zeta potential and particle size

During the 3-day incubation, I will measure the zeta potential and particle size of the transfection complexes. Zeta potential basically means the surface charge of the particle. Since the cell surface are slightly –vely charged due to proteogylcans in the cell membrane, the complex must be more positive to enter the cell. However it must not be too high, because if too much complex enter the cells, cytotoxicity can occur. Apart from that, the particle size is also measured. It cannot be too large as they need to enter through the nuclear pores which are only 10nm in diameter. Both can be measured using a nanozeta and size analyzer based on different conditions and pH.

Assays for transfection efficiency and cytotoxicity & Total protein measurement

After 3 days, I can then do my analysis. I have not learnt the MTT assay procedures which is done to measure the cell viability, so I will explain about the luciferase analysis assay which can consume the whole day(seriously). This is done to determine the transfection efficiency(how many cells take up the complex). The DNA that I used for the complex is a luciferase-encoding plasmid. Cells that have been transfected will have the luciferase gene and can express the luciferase protein. The cells are then washed and lysed using lysis solution and further scrapped using pipette tips. This must be done properly as it will greatly affect the results. I took 3.5 hrs to do 4 24-well plates.

Then, I will measure the light intensity by adding substrate to the luciferase protein in the sample using the luciferase analyzer machine. A BSA standard is also done to convert the light intensity value to protein concentration. Apart from that, total protein content is also done to determine what is the percentage of luciferase protein over the total protein expressed by the cells. Both the BSA standard and total protein content are measured using 96-well plate reader.

From the beginning to the end of the experiment, I do have lots of excel sheets and graphs to plot. I should say that the steps and procedures are quite tedious and time consuming. Yet, IBN is good place for students like us because they give you the freedom to conduct any experiments you want, so long as it is within the focus area of the lab.

As you can see I did not explain about the step-by-step procedures for each phase because it is very long, so if you want to know, just send me a comment. I will blog more about result analysis and siRNA on my next post.

A brief one on LMQA

A*STAR divisions especially IBN put great emphasis on safety in the labs. Almost everyday, I do receive emails on safety aspects and every month, I have to attend safety briefing. I have an RFID card that I must always carry with me, so that if there is any emergency occurence, they can track where I am electronically. Apart from that, each lab has a SOP and incident occurrence box at the entrance. This white box contains the necessary documents to operate the machines, and also filing of accidents that have happened in the labs. Every 2 weeks, a safety officer will regularly check the boxes if all documents are present. Plus, at every level there is a MSDS room where you can find all the A-Z details of the chemicals. We are also given a small card containing a list of safety contact officers in case if there is any accident occurrence.

Regards,
Nisha Bte Mohd Rafiq
0503254E
TG02