The St. Hilaire Lab studies important cardiovascular diseases such as Calcific Aortic Valve Disease (CAVD), Arterial Calcification due to Deficiency of CD73 (ACDC) and Medial Arterial Calcifica­tion (MAC). The similarity between all of these conditions is the calcification of cells, which leads to the main research question of the lab: how and why does a healthy cell transition into an osteogenic cell? In order to determine why a healthy cell becomes calcified, we perform ex­periments using both in vivo and in vitro environments. However, in order to begin in vitro experiments that involve valu­able human cells, basic techniques in cell culture such as plating and splitting are needed, and in vivo experiments require knowing the genotype of the animals used. With these fundamental techniques, biochemical analysis such as quantitative polymerase chain reaction (qPCR), west­ern blots, and staining can be performed in order to help answer research questions that pertain to the lab.

Introduction

Cardiovascular disease is the lead­ing cause of death in the United States, averaging around one in four deaths every year.¹ There are various types of heart disease and in many cases, it involves calcification of either arteries, valves or vessels. The St. Hilaire Lab focuses on the underlying cause of vascular disease, and more specifically, what drives vascular and valvular calcification in order to develop non-surgical methods to treat these conditions.² Calcific Aortic Valve Disease (CAVD) is the most common valvular heart disease and occurs when the leaflets in the valve become calcified, hardened and thick. This phenomenon occurs in around 30% of the aging population and covers a spectrum of conditions from aor­tic sclerosis (hardening of valve leaflets) all the way to aortic stenosis (blood flow to the aorta is blocked and results in heart failure).³ Currently, the treatment options for CAVD include surgical aortic valve replacement with either a mechanical or bioprosthetic valve. Mechanical valves are made from metallic alloys or plastic elements, whereas bioprosthetic valves are made from animal tissue. However, the lifespan of bioprosthetic valves is short and patients may require reoperation, indi­cating that surgical intervention is not the most effective option.⁴ Furthermore, the St. Hilaire Lab also studies Medial Arte­rial Calcification (MAC). This is a con­dition in which calcium deposits develop along the smooth muscle layer of the arterial wall, which can eventually destroy the vessel. MAC is often associated with diabetes mellitus, chronic kidney dis­ease, and aging.⁵ In addition to MAC and CAVD, the St. Hilaire Lab studies a rare disease called ACDC, which is Arterial Calcification due to a Deficiency of CD73. Research suggests that patients with this condition have certain inactivating mutations in the gene CD73. The CD73 gene breaks down extracellular AMP into adenosine. Without CD73 and adenosine present, patients develop vascular calci­fication and increased vessel tortuosity.⁶ The St. Hilaire Lab’s main goal is to un­derstand the mechanisms behind a healthy cell developing into an osteogenic cell as it occurs in these diseases. Understanding this phenomenon requires complex experiments and a strong foundation of the basic techniques used for such investiga­tions. These techniques include genotyp­ing and splitting/plating cells.

Methods

The St. Hilaire Lab research utilizes both in vivo and in vitro experiments. In vivo refers to working with living organisms such as mice, which are used for genotyping. In vitro refers to work that is performed outside of living organ­isms, such as cells. At the St. Hilaire Lab, different cells are used in cell culture, and this plays a vital role in the research conducted.

Genotyping

Genotyping is key for experiments as it determines the differences in genetic components by allowing us to compare the DNA of the sample to a reference sequence. It is especially important to help identify correlations between ge­netic variations and having abnormal/ normal phenotypes. At the St. Hilaire Lab, there are many different mouse lines that are used for genotyping, and these in­clude: TERT, TERT-Tg, MGP, CD73 and ERCC1/R26R. When genotyping TERT, MGP and CD73, the goal is to determine whether mice are knockout or wild-types. A knockout mouse is one whose DNA is genetically engineered to not express certain genes, whereas wild-type mice express the gene in a manner that is con­sidered to be normal and found in natural populations.

There are three main aspects to geno­typing: DNA extraction, polymerase chain reaction (PCR) and gel electrophoresis. As the first step, the DNA should be obtained from the mice samples. To do this, a small portion of the mouse’s tail is snipped and a certain amount of DNA lysis reagent as well as proteinase K is added to the tail to create a mixture. DNA lysis reagent acts as a buffer solution and breaks open cells to obtain DNA. Proteinase K inactivates nucleases or other substances that might degrade the DNA by digesting protein and removing contamination. After add­ing the mixture of DNA lysis reagent and proteinase K to each tail, the tubes are then put into a heat block, allowing for the denaturation of proteins and the actual extraction of DNA.

The next step in the genotyping process is PCR. PCR is comprised of three steps: denaturation, annealing and extension. The denaturation of the tem­plate is completed in the first step by DNA extraction in which the double stranded DNA is separated into single strands. An­nealing is the process whereupon primers, which are small molecules of DNA, bind to regions of the complementary single DNA strands. The final step is extension, in which the DNA polymerase extends the primer from the 3’ end all the way to the end of the amplicon. After these three steps, the target region of the DNA needed for observation is amplified.

After PCR, gel electrophoresis occurs to check whether PCR was successful and to determine the size of each DNA sam­ple. This is done by comparing the DNA samples to the DNA ladder, which indi­cates known base pair lengths of DNA.

In addition to genotyping, another aspect of the St. Hilaire Lab’s research is in vivo mouse dissections. Mice are quite similar to humans in anatomy, physiology, and genetics. Since the mouse genome is closely related to the human genome, using mice in genetic research is helpful for the study of different human conditions--in this case, CAVD and MAC. During these dissections, the main objec­tive is to remove the aorta of the mouse, which is the main artery that carries blood away from the heart and transfers it to the rest of the body. This is done so the cells from the aorta can be extracted and studied through performing experiments in cell culture. After the aorta is extracted, the tissues are sent to pathology to be put in paraffin blocks. Following this, there will be the opportunity to learn how to stain slides from these blocks.

Cell Culture

The St. Hilaire Lab works in vitro by experimenting with various kinds of cells. Cell culture is an important technique in molecular biology as it allows research­ers to study the biology, chemistry, and physiology of wild-type cells as well as diseased cells. Further, we had the oppor­tunity to learn methods in working with cells such as splitting, plating, and col­lecting cells. However, working with cells requires sterile technique. It is essential to spray everything with ethanol before bringing those items under the chemical hood, as this will prevent bacterial and fungal contamination of the cells.

To split cells, it is essential to look at the confluency, which is the percentage of the surface of a plate or dish that contains cells. If the cells are around 90% conflu­ent, they are ready to be split. If cells are not split in time, they may grow on top of each other or stop growing altogether. When cells grow on top of each other, the cells at the bottom have little access to nutrients, causing them to die, while the ones on top may detach and float. To split cells, the media (containing nutrients for the cells) and trypsin (an enzyme that breaks down proteins that enable cells to stick to the vessel) are placed in the water bath to warm them. After the media and trypsin are warmed to room temperature, the process starts by aspirating the old me­dia and washing the cells with Phosphate Buffered Saline (PBS) twice. PBS is used opposed to water as it prevents cells from either rupturing or shrinking due to os­mosis. Next, the trypsin is added, and the plate is left to incubate for a few minutes. Once the plate is done incubating, the cells lift off the plate, which can be seen with the microscope, suggesting that they are ready to be split into separate wells or plates. Before they are split into separate wells/plates, media is added to neutralize the trypsin reaction.

Plating cells is important for future experiments. In order to plate cells, the number of cells is counted using a ma­chine and then split evenly into a six-well plate to use for future experiments. If there are any remaining cells, they are mixed with a freezing solution and restored in the liquid nitrogen tank for the next use. Splitting and plating cells are significant for following experiments such as qPCR, western blots, and staining.

qPCR is utilized for determining the actual amount of PCR product that is present in each cycle. The process uses a fluorescent report in the PCR reaction, al­lowing measurement of DNA generation. The main difference between PCR and qPCR is that PCR is a qualitative tech­nique that shows the absence or presence of DNA, whereas qPCR is a quantitative technique that determines the amount of DNA amplified after each cycle.

Western blotting is a method that detects a target protein within a tissue sample or lysate. The protein molecules are separated by size through gel electro­phoresis. Gel electrophoresis works by loading DNA into wells within the gel and an electric current causes the negatively charged DNA fragments to move towards the positively charged electrode. Western blotting is a method of gel electrophoresis; however, it deals with proteins as opposed to DNA.

Lastly, cell staining provides a clear­er visual of cells and their components under a microscope. Cell components are stained differently to allow for compari­son depending on what the researcher is studying. For example, at the St. Hilaire Lab, cells have been stained using healthy cell markers, osteogenic cell markers and intermediate markers, which indicate the process of a healthy cell undergoing transition into an osteogenic cell. This allows for clear visualization of the cell becom­ing osteogenic. These learning techniques, such as genotyping and the basis of cell culture, can be utilized for projects that will involve qPCR, western blots and staining in order to study how a healthy cell transforms into a calcifying cell.

Results

After gel electrophoresis, the mice samples can be genotyped. ERCC1 is a gene that is genotyped as either wild-type or flox. Flox refers to the sandwiching of a DNA sequence by two loxP sites. This utilizes the Cre-loxP system, where Cre is a protein that can catalyze the recom­bination of DNA between specific sites. These sites are the loxP sequences, which contain specific binding sites for Cre. Cre will then get rid of the loxP sites so that the DNA sequence in between those sites can be translated.

Figure 1: ERCC1 genotyping results. Both + labels indicate that the mouse is wild-type; one + and one f label indicates that it has one loxP site; two f labels indicate that there are two loxP sites. The positive control represents the allele as flox and the negative control represents the allele as wild-type. The no template control at the end is water and it is important for detecting contamina­tion or the lack of amplification. 

The gene R26R works similarly to ERCC1. The mutant gene contains loxP sites, which surround a specific part of the DNA sequence. However, the stop sequence after the loxP site contains the enhanced yellow fluorescent protein gene that is expressed with the implementation of the Cre-loxP system. With the addition of Cre removing the loxP sites, the stop sequence is deleted, and the yellow fluo­rescent protein is expressed in the mutant gene.

Figure 2: R26R genotyping results for the same mice samples as ERCCI. Both + labels indicate that the mouse is wild-type; one + and one Y label indicates that it has one loxP site; having two Y labels indicates that there are two loxP sites. The positive control represents the allele as Y and the negative control represents the allele as wild-type. The no temple control at the end is water.

Figure 3: TERT-Tg genotyping results. Unlike TERT, MGP, CD73, andERCC1/R26R, this gene has only one possibility in genotyping; it is a sim­ple +, confirming that the mouse is transgenic. The positive control represents the allele as transgenic, and it includes the internal control. The negative control represents the allele as the internal con­trol. The no template control at the end is water.

Tert-Tg is a line that is transgenic, meaning that the mouse has been genet­ically engineered with an extra piece of DNA added to its genome. When geno­typing this line, there is only one geno­type that can occur. It is expected to see a band at both 200 base pairs and 600 base pairs. The first band at 200 base pairs is an internal control that confirms the PCR ran successfully. The band at 600 base pairs indicates that the mouse is transgenic.

Figure 4: TERT genotyping results. The positive control represents the allele as knockout and the negative control represents the allele as wild-type. The no template control at the end is water.

TERT, CD73 and MGP are mouse lines that are all genotyped the same way. For example, +/+ indicates that the mouse is a wild-type; +/- indicates that the mouse is heterozygous (one wild-type allele and one mutated allele); -/- indicates that the mouse is a knockout.

In vitro experiment results and techniques involve taking pictures from the microscope to track how cells are doing. It is important to take pictures before splitting the cells in order to ensure they are confluent enough. It is just as crucial to keep checking the cells even after a day or so following splitting them to make sure the cells are growing properly and have enough nutrients to do so.

Figure 5: Cells are around 90% confluent and ready to split.

The picture above shows HEK 293 cells, which are human embryonic kidney cells. These cells are around 90% conflu­ent and are ready to be split. The picture below shows the cells one day after split­ting them. The cells are much less conflu­ent compared to the picture above.

Figure 6: One day after the cells have been split; the cells are less confluent and have more space for nutrients to grow.

Conclusion

Both genotyping and cell culture techniques such as splitting, plating, and collecting cells are very important for future complex experiments. Genotyping helps researchers understand the heredity behind an organism’s genome. By looking at band sizes, it can be determined if the organism contains a knockout gene, is wild-type or is heterozygous for that spe­cific gene. This information can help discover whether the parents of the organism are either heterozygous or homozygous for the gene as well. Furthermore, geno­typing allows researchers to see if there is a break or lesion in the DNA and how that will have an impact on the organism, such as having a disease. On the other hand, cell culture helps researchers understand the biology, physiology, and chemistry behind cells. Sterile practice is vital in cell culture to ensure that bacterial/fungal contamination does not occur, which will greatly impact results. Practices such as spraying objects and media with ethanol before bringing them under the chemical hood will create the most sterile environ­ment we can produce. Basic techniques in cell culture can produce opportunities to perform qPCR, staining, and western blot experiments, which can be used to study how a healthy cell transitions to a calci­fied cell.

 

 

Bibliography

Losurdo, Fabrizio, et al. "Medial Artery Calcification: The Silent Killer of the Leg." American College of Cardiology.

Yutzey, Katherine E., et al. "Calcific Aortic Valve Disease." American Heart Association Journals, vol. 34, no. 11, 4 Sept. 2014.

“Heart Disease Facts.” Centers for Disease Control and Prevention, Centers for Disease Control and Prevention, 14 Oct. 2022, https://www.cdc.gov/heartdisease/facts.htm.

 

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