Week 2: Transfection, Exploration, and Caturday!
Last week I left off discussing my acclimation to Inuyama and a brief overview of functional analysis. This week, my cell culture practice continued in the molecular biology laboratory here at the PRI. We are using HEK293T cells, a type of cell widely used in research because they grow reliably in external conditions and work well for transfection, which in our case, is the process of introducing the TAS2R38 gene into the cells. The HEK293T cells are grown in a petri dish with Dulbecco’s Modified Eagle’s medium (DMEM) and 10% fetal bovine serum (FBS). Think of these as the cells’ nutrients. DMEM contains glucose sugars, amino acids, and vitamins, all of which are necessary for healthy cells. It also contains phenol red, which is used as a pH indicator. DMEM begins red in color, and gradually turns yellow as the pH drops below 6.8. A stable pH (close to 7) is necessary for cell cultivation, and the cells are stored in a CO2 incubator to control the pH value of the media. The fetal bovine serum contains many growth factors and proteins that allow cultured cells to grow and divide in a laboratory environment. Media changes are performed every 2 days for about 20 days to provide fresh nutrients to the cell line. After the cell spread in the dish is around 80% covered, the cells are ready for transfection.
Tuesday, I began transfection using 3 Japanese macaque (Macaca fuscata) haplotype samples for the TAS2R38 receptor gene (Mf-A, Mf-B, and Mf-O), as well as a human haplotype (hT2r38). Mf-A and Mf-B haplotypes are found at high frequency among many Japanese macaque populations. The Mf-A haplotype seems to have a greater sensitivity to PTC than Mf-B (Hashido et al. 2015). The Mf-O haplotype is mainly found in the Shimokita (SMK) population in Northern Japan. More data are necessary to assess the Mf-O haplotype’s sensitivity to PTC. In my next blog, I will go through the genetic differences between these 3 haplotypes, but for now let’s overview the transfection protocol.
To artificially introduce the TAS2R38 DNA into the cells, we first need to clone the DNA to increase the number of copies available. To do this, the gene is inserted into E. coli bacteria DNA. E. coli have very simple, circular DNA (called a ‘plasmid’) that can replicate very quickly. These plasmids have specific sites where you can cut, copy, and paste a new gene into the circular DNA (we use the pEAK10 plasmid). Then, the natural mechanisms of DNA replication do most of the work, resulting in many E. coli cells with recombinant DNA, or DNA from both the E. coli and our foreign TAS2R38 gene. We then purify the DNA, removing proteins and other cellular components. The end result is called a vector, which consists of many copies of our TAS2R38 DNA, and is now ready for transfection.
A mixture of optiMEM and Lipofectamine is prepared and added to another mixture containing our vector and G protein. OptiMEM and Lipofectamine are two reagents used to increase the transfection efficiency of the plasmid DNA into the cells. After all of these components are combined, they are added to a 6 well plate and the HEK392T cells are transfected with the TAS2R38 gene haplotypes. After a 6-hour incubation period, each sample mixture is transferred to a 96 well plate to prepare for the final stages of data collection, calcium imaging, the next day.
Nagoya is the largest city in the Chubu region of Japan and is located about 30 minutes from Inuyama. Last weekend Mita-san and I visited the Nagoya Port Aquarium, located on one of the largest container ports in Japan. Many cruise ships visit Japan through this port, and it has recently been redeveloped as a leisure district. It was a beautiful day with the sea breeze and sun shining as we entered the aquarium. Nagoya Port Aquarium is home to many creatures, including orca whales and bottlenose dolphins! This aquarium is one of the few with killer whales, and I was mesmerized by their beauty. The dolphins were equally as fascinating, and the baby dolphin was so kawaii (cute)! One creature I have never seen before was the gigantic Japanese spider crab (Macrocheira kaempfer). It is found along the southern coasts of the Japanese island Honshu, and fully grown it can reach a leg span up to 15 feet long! Between the five aquatic landscapes represented at the aquarium, we spent a good part of the day there. For a late lunch, Mita-san and I tried a regional specialty of Nagoya, misokatsu. Breaded, deep-fried, and covered in a savory miso sauce, this pork dish is my favorite meal (so far) during my stay in Japan. Pictured below, it was served with rice, miso soup, and salad with ginger dressing. After lunch, we headed to the downtown district called Sakae for shopping and sight-seeing. There were many people outside enjoying the day throughout the district. One of the most fascinating things I saw while in Sakae was Oasis 21. The building contained underground stores and restaurants, but the most fascinating characteristic was the roof. It is a large oval glass structure that floats above the ground level and is filled with water. Effectively cooling down the temperature of the shopping area below, the water also makes a lovely visual effect. Walking onto the roof and watching day transition into night is a memory I will cherish forever.
This weekend I went back to Nagoya and sought out another delicious regional dish, misonikomi (udon noodles in miso soup), at the noodle shop Yamamotoya. The dish was brought out in a traditional earthen clay plot, and the soup was still sizzling as it made its way to my table. One then uses a traditional bamboo spice shaker (20 inches long!) to add a dash of red pepper from one end of the shaker or a dash of a special “seven spice” blend from the other end. As a soup lover, misonikomi was amazing, and I now know why Yamamotoya has been successful for nearly a century.
Satisfied and full, I headed to Cats Gallery, a cat café in Nagoya to get out of the sun and relax. What is a cat café you ask? It is a themed café where customers can pet and play with cats! Many apartment complexes in Japan do not allow pets, so this is a great place to enjoy some time with these wonderful animals. Cats Gallery has around thirteen cats. From playing with the active individuals, to petting those taking a “cat nap,” visiting Cats Gallery was a relaxing stop in my Nagoya adventure and a must-see attraction while visiting Japan.
Week #2 had many adventures, both in the science and cultural realms. I am thoroughly enjoying my stay here in Inuyama. In the week ahead I will continue functional analysis on the TAS2R38 gene for Japanese macaques, and then take a weekend trip to Osaka. Stay tuned for more exciting news from half way across the world. Thank you for tuning in for another week of my internship experiences! Check this website next week for blog post #3!