Reflections

In 1963, Sydney Brenner discovered that the tiny, soil roundworm, Caenorhabditis elegans could be a powerful model system in the scientific community. The “worm”, as affectionately called in our lab, has a constant 959 cells in its body after exactly 131 of the 1090 cells die during development. In addition, its genome shares 40% homology to humans and a fast lifecycle with a high brood size facilitates short-time scale studies that have implications toward mammals and humans. These properties allowed developmental cell death pathways and neural development to be extensively studied.

Since then, the c. elegans’ complete cell lineage of all differentiated cells has been mapped making it possible to know exactly how any cell was derived. A comprehensive pathway of programmed cell death, or apoptosis was identified and applied to humans. Interestingly the results from development and apoptosis research using c. elegans allowed new understandings of various proteins and mechanisms in cells, arguably setting the course of developmental biology for the next five decades.

Our lab is dedicated to researching these classic worm studies like apoptosis related effects, and developmental cues and gradients on a molecular and genetic level. Certain projects involve identifying the genes regulating cell determination and differentiation, while others aim to inspect cell fate and reprogramming of a fully developed cell type (a unique and exciting stem-cell biology spin off). The project I have the opportunity to work on nicely blends apoptosis and development to understand the molecular mechanisms ensuring inheritance of healthy mitochondrial DNA.

At the core of the project lies the immortality of genetic information. We, living bodies, are simply messengers or information carriers, alive for enough time to pass on genetic information by egg and sperm through our germline. This tissue producing egg and sperm, the germline, then is an immortal lineage and the only thing connecting us to our ancestors and our ancestors to the future little ones to come. I am investigating how the germline is sustained throughout so many generational transitions, and over such long a time scale, with a focus on mitochondrial DNA (mtDNA) inheritance. We hypothesize germline apoptosis is a crucial step in ensuring preferential passage of healthy mtDNA molecules. In order to understand mtDNA transmission through the germline, I am working on developing a preliminary selection condition, crossing various strains to produce a unique desired genotype, and constructing a visualization method for mitochondria in early embryos. The results of these projects will offer the scientific community a better understanding of mtDNA selection pressures during inheritance, but have offered me both more knowledge of biological techniques and concepts, as well as an appreciation for the patience, persistence, critical thinking and analysis skills required as an effective researcher.

A short term goal reached over this summer was doing a genetic cross to create a double mutant strain. I crossed a strain of C. elegans that had a point mutation on the nuclear gene, ced-3, with a mtDNA mutant containing a 3.1 kbp deletion in the mitochondrial DNA. Since mtDNA is inherited maternally, whereas nuclear DNA is inherited from mother and father, we utilized two different inheritance modes and systematically planned out crossing these two strains to obtain a desired genotype. To confirm the presence of both mutations after the cross, we conducted PCR (polymerase chain reaction, which amplifies a specific area of DNA), and visualized the presence of the deletion and ced-3 gene using another common lab technique, gel electrophoresis.

In addition, I began the exciting yet tedious journey of molecular cloning to create a GFP fusion construct. Ideally, we aim to visualize mitochondria through a fluorescent protein in the germline and during the early embryo stage using an introduced genetically engineered plasmid construct. And although the project is still in its preliminary stages, I’ve isolated the insert containing DNA of interest, and ligated that to a vector backbone containing a unique promoter to drive the genes. I learned how to conduct restriction digests to isolate a specific sequence of DNA. I also became familiar with transformations, which introduce DNA into E. coli bacteria for amplification. And I also refined my skills in running gels, visualizing and extracting bands for sequencing and further digests.

Contrasting from our “macroscopic” world, where, for example, the amount of raisins added in oatmeal raisin cookie recipes can vary, but the final product is the same delicious dessert, the “microscopic” world of DNA is very tightly regulated. During PCR, measurements a few microliters off could potentially yield no product – no amplified DNA. Or, during transformations, if a water bath is not calibrated to the exact 42 degree temperature, in just 30 seconds, the cells will not be fit to uptake DNA and the entire transformation could potentially be void. I’ve learned to be careful in my calculations, and frugal in my volumes used. Many times I’ve seen unexpected results and bands of surprising fragments lengths, which lead me to backtrack and analyze possible areas of technical error, or alternate biological explanations. Each erroneous result, though at times painful, reminds me how blindfolded we are to the polymerases, nucleotides, and protein interactions that compose living systems, but also how refining my experiments is possible for future successes.

This summer, with lab work, writing workshops, skills seminars and GRIT lecture series, has been a inspiring, exciting and stimulating one! I’m grateful to my mentor and faculty advisor for guidance and encouragement, and thankful to CSEP coordinators and The Arnold and Mabel Beckman Foundation for the Beckman Award, which has allowed me to participate in this tremendous opportunity. I’m excited to apply what I’ve learned and observed from the presentations and workshops and to continue researching and growing intellectually as I spend time in the lab.