Week 2:
Carver College of Medicine

June 3, 2014

This week started with some subcloning projects. This is a commonly used technique in molecular biology to insert a specific gene from its parent plasmid to the destination plasmid to study its genetic effect on bacteria. The process of subcloning involved the excise of the gene of interest (the insert) by restriction enzymes, which was usually purified using gel isolation. Then PCR (polymerase chain reaction) was used to multiply the number of copies of the insert. The same restriction enzymes were also used to cut the destination plasmid to create complementary sticky ends for ligase to recognize. CIAP (calf-intestinal alkaline phosphatase) was also added to prevent self-ligation of the plasmid themselves. The digested destination plasmid was also purified prior to use. Then DNA ligase was used to connect the insert and the destination plasmid. The molar amount of the insert was always in excess to inhibit the self-ligation of the destination plasmids. The obtained plasmids were then transformed into E. coli to replicate the number of plasmids along with the growth of the E. coli colonies. In addition, in order to grow only the transformed E. coli which contained the desired plasmid and inhibit the growth of undesired bacteria, a marker gene was also added to the destination plasmid. We chose the marker gene to be resistant to the antibiotic ampicillin. Therefore, the E. coli that were transformed would also pick up the marker gene and became ampicillin-resistant. They could grow in cell culture media that contained ampicillin whereas the untransformed E. coli could not and therefore died out. Finally, I used miniprep (process described later) to harvest the plasmid DNA and used them to transfect rat embryo hippocampus neurons, which were the subject of our model for stroke caused by mitochondrial fission and fusion events. We did a total of eight transfections with eight different insert genes. We treated differently-transfected neurons with 15 minutes of oxygen/glucose depravation to mimic what happens during stroke. The depravation of oxygen in cell culture media involved the deoxygenation process with nitrogen gas similar to running an air-sensitive chemical reaction.

Another major project of this week was the LDH cytotoxicity assay. Lactate dehydrogenase (LDH) is a cytosolic enzyme present in many different cell types. Plasma membrane damage releases LDH into the cell culture media. Extracellular LDH in the media can be quantified by a coupled enzymatic reaction in which LDH catalyzes the conversion of lactate to pyruvate via NAD+ reduction to NADH. Diaphorase then uses NADH to reduce a tetrazolium salt (INT) to a red formazan product that can be measured at 490 nm. The level of formazan formation is directly proportional to the amount of LDH released into the media, which is indicative of cytotoxicity. I will describe here the experimental procedure concisely. When I received the cell cultures treated with different chemical compounds, I first sucked the culture media up using aspirator, washed the wells with DPBS (Dulbecco’s Phosphate Buffered Saline), and sucked the wash solution up again with aspirator. Then I combined 10 µL of each cell media (which contains LDH) and 20 µL of assay buffer and measured the absorbance at 490 nm using a luminometer. After incubating at room temperature for ten minutes, I added 20 µL of stop buffer to each tube, vortexed, and again measured the absorbance. We will do the statistical analysis next week.