LECTURE: GENETICS

HOW GENES CAN INFLUENCE LIFESPAN

● Anti-longevity gene: contributes to a shortened lifespan
○ When deleted/mutated, lifespan increases
● Pro-longevity gene: contributes to a lengthened lifespan
○ When deleted/mutated, lifespan decreases

GENOMIC DATABASES
● Modern genome sequencing has made almost any genetic sequence available at our fingertips
● Geneticists can use genomic databases to probe questions about the similarity of genes
between organisms
● Many databases available – some broad spanning (e.g. NCBI Gene), others organism-specific
(e.g. FlyBase)

ANALYZING SEQUENCES
● Bioinformatics: interdisciplinary field that uses computational methods to gain insights into
biology through the analysis of biological data
● You can use databases and bioinformatics tools to investigate:
○ The similarity of genes and proteins between organisms (conservation)
○ The differences that distinguish them (mutation, divergence, evolution)
○ Structure/function relationships

COMPARING GENES
● Genes in different organisms that are similar in sequence and that perform the same function
are said to be homologous
● Organisms share a common ancestor
● Sequence is conserved despite speciation to maintain function

Protein sequences are more conserved than DNA sequences

● DNA sequence varies more than protein sequence
● Protein sequence appears more conserved
● Multiple codons can code for the same amino acid
○ The wobble effect

TOOLS WE USED IN LAB

● BLAST: Basic Local Alignment search tool
● Aligning sequences with clustal omega

ALIGNMENT ANALYSIS
● You can align your query sequence with other sequences to look for regions of conservation
● A good alignment depends on how similar the tested sequences are
● Regions of conservation may indicate important areas within a protein that relate to its function
or structure
● Sequence conservation: where protein sequences are the same across different organisms

WORLDWIDE LONGEVITY
Blue zones: Populations of people with a higher than average lifespan
● Individuals in these areas have similar environment and lifestyle characteristics
○ No smoking, exercise, eat nuts/beans/turmeric, sunshine, etc.,
● Genetics plays a small role in aging, with 7% of the diversity in longevity explained by genetic
factors
● changes in our genome stability correlate with the aging process

CELL BIO LAB

● Working with immortalized (replicate indefinitely) and adherent (attach to the surface of the
container) mouse myoblast cell line named C2C12
● The cell culture media we use for our cell line is called Dulbecco’s Modified Eagle’s Medium
(DMEM) which is supplemented with 10% Fetal Bovine Serum (FBS)
● FBS is a serum supplement for the cell culture and contains growth factors and other biological
components that the cells need
● DMEM and add FBS = “supplemented cell culture media.”
● These dishes of cells are stored in an incubator which regulates the environmental temperature
and CO2
● The C2C12 cell line is maintained at 37°C with 5% CO2
● We control the sterility of our environment by using a biological safety cabinet (BSC)
● The BSC is specially designed to control air flow to prevent unfiltered air from the outside
environment from entering the working space inside the cabinet
● This is achieved by an air curtain that blows air across the opening of the BSC
● The air in the BSC undergoes specialized filtration using a High-Efficiency Particulate Air (HEPA)
filter to ensure that the air is as clean as possible inside the BSC
● A fume hood is meant to protect the user from the chemicals/reagents within the fume hood. A
biosafety cabinet is meant to protect the biological agents inside it from the contaminants
surrounding them in the air

Function of materials:
● PBS: Washing cells and removing residual CCM when passaging cells
● Un-supplemented DHMEM: Cell Culture Media (no FBS). Used when FBS isn't required
● Supplemented DMEM Cell Culture Media: Cell Culture Media (CCM) that has been
supplemented with 10% Fetal Bovine Serum (FBS) and antibiotics
● Trypsin: Detach cells from plate when passaging
 Lab Part 1: Introduction to Cell Culture Techniques
● Purpose: To observe cell morphology and estimate confluence in cell cultures using an inverted
compound light microscope, To practice passaging (splitting) cells using aseptic technique which
is required to maintain healthy cell cultures
● Supplemented DMEM cell culture media (sup-DMEM), unsupplemented DMEM (unsup-DMEM),
phosphate buffered saline (PBS), and trypsinase enzyme (trypsin) in the incubator to bring to the
same temperature as the cells
● Spray down your gloved hands and the BSC with 70% EtOH and disinfect the workspace
● Spray down anything that goes into the BSC with 70% alcohol, disinfecting all supplies as you
move them into the BSC

Lab Protocol 2: Passaging C2C12 Cells

1. Remove one cell culture plate containing the C2C12 cells from the incubator and examine under
the inverted microscope. Observe the cell morphology and estimate % confluence
2. Spray the outside surface of the C2C12 plate with 70% EtOH and place in the BSC.
3. Remove the sup-DMEM, unsup-DMEM, PBS and trypsin from the incubator, spray with 70%
EtOH, and place in the BSC.
4. Label two new 60 mm cell culture dishes and a 1.5 ml tube (with the lid closed) with:
1. the date the cells were divided
2. passage number (e.g. if the starting dish with the C2C12 cells is P3, label the dish for the
next passage P4)
3. the type of cell line being used (C2C12).
4. Your (or your group’s) name.
5. Connect a P1000 pipette tip to the aspirator pump tubing. Be careful this tip does not ever come
into contact with the BSC surface. If it does, put the contaminated tip in the waste container and
replace it with a fresh one.
6. Tilt the C2C12 plate so the media accumulates along the bottom, and use the aspirator pump to
remove the old cell culture media from the dish.
7. Use the P1000 (set to 1000 µl) to add 1 ml of PBS to the plate. Swirl and aspirate.
8. Use the P1000 (set to 1000 µl) to add 1 ml of PBS to the plate. Swirl and aspirate.
9. Use the P1000 (set to 1000 µl) to add 1 ml of trypsin to the plate. Swirl across the surface. Place
in 37°C incubator for 5-7 minutes.
10. Remove the plate containing the trypsin from the incubator and view under the inverted
microscope. Tap the dish and check if cells are adherent. If still adherent, place back in
incubator for another minute
11. After cells have detached, spray the C2C12 plate with 70% EtOH and place in the BSC.
12. Use the P1000 (set to 500 µl) to add 0.5 ml of Supp-DMEM to the dish. Swirl the media around
the dish (clockwise x3, counter-clockwise x3, forward and back x3, left and right x3).
13. Tilt the dish so the cell suspension accumulates along the bottom edge, and use the P1000 to
pipette the cell suspension up and down 3X to break up any cell clumps. The dish will have a
total of 1.5 ml of cell suspension.
14. Use the P1000 (set to 1000 µl) to transfer the 1.5 ml of cell suspension into the labelled 1.5 ml
tube. It will take two transfers to move all of the cell suspension.
15. Split the cells based on the confluence you estimated above. For the C2C12 line, the following
works well to achieve a 70-90% confluent dish in 3 days (these values are using the 1.5 ml of cell
suspension in your tube and a petri dish that holds 5 ml):
● 30% confluent – split 1:5
● 0.3 ml of cell suspension + 4.7 ml of unsupp-DMEM
● 70% confluent or greater – split 1:10
● 0.15 ml of cell suspension + 4.85 ml of unsupp-DMEM
1. Add the required volume of unsupp-DMEM to the two new dishes using a serological pipette
and the green Pipet Pump.
2. Use the P200 to add the required volume of cell suspension to each of the newly labelled cell
culture plates. Swirl (forward and back x3, left and right x3). Repeat for the second plate.
3. Place both new plates in the 37°C incubator. New cell growth should be observed after a couple
days.
4. Clean up: all waste (tips, pipettes) in waste container inside BSC, old TC dishes go in container
with 10% bleach (in fumehood), spray down BSC with 70% EtOH.
5. Ensure the 1.5 ml tube containing the cell suspension is closed, spray with 70% EtOH, and this
tube can now be removed from the BSC and taken to the bench to be used for a cell count using
a haemocytometer.

Lab Part 2: Performing Cell Counts with a Haemocytometer

● Count cells in corners and centre square

● Dead cells appear blue

1. Spray the haemocytometer with 70% alcohol and wipe with a KimWipe. Wait for the alcohol to
evaporate (~1-2 minutes)
2. Place a coverslip on the haemocytometer.
3. Label a 0.2 ml tube as CS-T (=cell suspension + trypan blue)
4. Tap the microcentrifuge tube containing your cell suspension to re-suspend cells that have
settled to the bottom of the tube. You can also gently pipette the solution up and down within a
pipette tip to re-suspend cells.
5. Immediately after tapping the cell suspension tube, transfer a 50 µl aliquot of cell suspension
into the 0.2 ml tube labelled CS-T
6. Transfer a 50 µl aliquot of 0.4% trypan blue into the CS-T tube. Gently invert the tube 2-3 times
to mix.
1. Review figure 5. Tap the 0.2 ml tube (CS-T) to re-suspend the cells. Immediately after tapping,
use the P20 pipette to take a 12 µl aliquot of the cell suspension/trypan blue solution from the
CS-T tube and apply to the haemocytometer as demonstrated in the video and figure 5. Very
gently fill ONE chamber underneath the coverslip, allowing the cell suspension to be drawn out
by capillary action. Do not over-fill or under-fill the chamber.
2. Repeat step 7 to fill the chamber on the other side of the haemocytometer.
3. Place the haemocytometer on the microscope stage. Starting with the lowest objective, bring
the 3 x 3 grid into focus. Adjust the light intensity and condenser diaphragm until you can see
this grid and cells contained within.
4. Switch to the 10X objective and focus on one of the squares (i.e. focus on one of the red squares
from figure 5c).
5. For each of the 5 red squares, count the # of live cells (appear clear/translucent) and dead cells
(stained blue) and record your data in table I. Use the cell counter to keep track of the counts.
Note: the top boundary and left boundary of each square should be counted – the right and
bottom boundaries should not be counted.
6. To accurately count cells, there should not be too many cells that they overlap. A general rule is
no more than 50 cells per red square.
7. If there are too many cells, dilute the cell suspension with the culture media, and then repeat
from steps 5 onward using the diluted cell suspension. Try a 4-fold dilution (your initial 1:1 ratio
of cells and trypan blue is a 2-fold dilution)
8. Each student should make their own cell counts and record the data in table I.
9. When you are finished, clean the haemocytometer and coverslip with 70% EtOH, gently wipe
with a KimWipe, and leave it on your bench to dry.

Complete steps 7 – 10 within 20 minutes, as trypan blue is toxic to cells and will eventually enter
and kill the viable cells.