Manipulation Measure

DNA Cre Lox, CRISPR, Cloning SNPs

Isolate with gel electrophoresis
PCR
GWAS (genome mapping)

RNA RNAi Isolate proteins (gel electrophoresis)

PCR (old PCR)
Microarrays

Protein Antagonists, Agonists, Isolate proteins (SDS Page), Centrifugation

Antibodies, Denature Western blot
ELIZA
Immunohistochemistry (IHC/IF)
Chromatography

Behavior Optogenetics, granting access Frequency, Duration

or restricting Latency
Light-Dark box
Open field arena
Intruder test
Social novelty test
Food Intake

CreLox
https://www.jax.org/news-and-insights/jax-blog/2011/september/cre-lox-breeding-for-dummies

CRISPR
Gene Knock In
● One-for- one substitution of DNA at a locus or inserting a sequence not in the locus
● Mostly experimented with immunodeficiency and stem cell research
Knock-Out
● Stop specific genes from fulfilling their functions, they can be used to study the impact of a
specific gene on the body/behavior

Proteins
● Centrifugation- ​mass and density
■ Differential Centrifugation
● Separates based on Density
● Multiple steps at increasing speeds removing pellet or supernatant
■ Rate Zonal Centrifugation
● Separates based on mass/shape
● Spins for period of time and proteins separated into bands
● Gel Electrophoresis
○ Separate based on charge, mass, and 3D shape
○ SDS-Page
■ Type of gel electrophoresis uses negatively charges detergent SDS and heat to
denature proteins and breakup complexes
■ Proteins separate based on size
● Liquid Chromatography
○ Proteins separate based on mass, charge, or binding affinity
■ Gel Filtration (Size)
● Beads have depression where small proteins move slower through, larger
proteins exit first
■ Ion-Exchange (Charge)
● Beads are + or - and + beads will hold onto - proteins, then remove
bound proteins by increase concentrations of NaCl
● Proteins with a weaka net - charge will come off the + beads with low
salt
● Stronger attractions require more salt
■ Affinity (attraction to a specific ligand molecule)
● Ligand molecules bind to the protein with the chemically attached to the
beads- only those with affinity will stick, remove the bound proteins by
adding salt or low pH
○ Limitations- Reliance of polarity can result in similar compounds exiting at the same time
aka coelution, with binding to the bands some molecules may bind so strongly they are
never release/measured
Antibody-Dependent
● ImmunoHistochemistry
○ Antibodies contain fluorescent molecules- detects where proteins are located
● Western Blot (Immunoblot)
○ Separates Proteins via SDS-Page, transfer proteins onto a nitrocellulose membrane, dd
antibiotic specific to protein of interest, add second antibody (specific for the 1st antibody
 and contains enzyme), add colorless substrate (enzyme will convert it to a colored
product
■ Limitations- MUST have the antibodies available especially as many antibodies
will bind to other proteins, not just the desired one, tissue prep is specific to the
western blot
● ELISA (Enzyme linked immunosorbent assay)
○ Antibody is added to plastic well, protein mixture added (protein recognized by the
antibody will bind to well), second antibody that recognized same protein is added
(contains an enzyme that makes product colored)
○ The more color produced, the more original protein in the mixture
■ Indirect​: the protein sample is bound through adsorption, directly
(and non-specifically) to the well. Next, an antibody is used to detect
the presence of one of the proteins contained in the sample, known as
the antigen.
■ Sandwich​: a 'capture' antibody is bound to the well first, and when
the sample is added, only proteins the antibody recognizes are
'captured'. Next, a second 'detection' antibody is used to detect the
bound protein. The 'capture' and 'detection' antibodies are commonly
called 'matched-pairs'. Finally, a third, enzyme-labeled antibody is
added to detect the 'detection' antibody.
■ Competitive​: a primary antibody is incubated with the sample, which
forms a complex. The complex is then adsorbed to the wells. Next, a
secondary antibody is added to the wells, which recognizes the
primary antibody only if it is not bound to the antigen. Therefore, the
secondary antibody 'competes' with the antigen.

Purifying
Flow cytometry
● Fluorescence-activated cell sorter (FACS)
○ Single cells moved through a laser where the scattered light (forward, side) separates the
cells based on a chosen proterites
○ Separated cells can be grown by themselves
○ Label cell of interest with fluorescent dye and laser detects those with the dye
Laser Capture Microscopy
● Thin section of tissue, using microscope find cells to isolate, in the machine a laser will melt a
small plastic probe that is then touched to the cell of interest, once cooled the probe is lifted with
the cell
Light Microscopy
● Bright Field
○ Eyes detect what light gets through the sample and what doesn’t
■ Cell fixation
● Treating the cell to maintain structural integrity
● Phase Contrast Microscopy and differential interference contrast (DIC) microscopy
○ Unstained, live cells
 ○ Uses differences in thickness and refractive indices within the cell to enhance contract
■ P-C→ thin specimen
■ DIC→ thicker specimen
○ Limitations- limited resolution, lower magnification, poorer surface view of the
specimen- never be as good as an electron microscope
Fluorescence Microscopy- easy detection of protein/antigen of interest
● Absorb light at one wavelength and emit light at a different wavelengths
Confocal Microscopy
● Uses lasers to observe thin sections of a thick sample
● Limitations- fluorescence wears off, possibility of photobleaching

Types of microscopes
● Electron microscopy
○ Scanning EM
○ Surface Topography
○ 2-Photon Microscopy

Neuroimaging

Structural
● Pneumoencephalography
○ Can see injures in ventricular space but mega painful
● Cerebral Angiography
○ Catheter inserts contrast showing blood vessels in the brain
● Computerized Axial Tomography (CAT, CT)
Image
● PET scans
○ Radioactive dye measuring blood flow, oxygen use, and glucose metabolism to evaluate
bodily functions, good for detecting cancers
○ Limitations- Radioactive dye, bad for people with diabetes
● MRI
○ Uses magnetic field to create images of organs, good for brain bleeds, etc
○ Limitations- can not be used on people with metal in them such as pacemakers
● CT
○ Series of beams through the body to create an image
○ Limitations- really still, low levels of radiation

Cell Proliferation
● BrdU
 ○ Cells are treated with BrdU, a thymidine analog incorporated into DNA during cell
proliferation, detected with a special antibody= high rates of BrdU correlate with high
cell proliferation rates
■ Limitations- toxic over time
● Protein Proliferations
○ PCNA
○ Detects cells in the late G1 phase and S phase
○ Prognostic value in cancers

Stemness Properties
● Test expression of specific markers by qPCR or Western blot responsible for stem-like qualities
● Self-renewal
● Multilineage potential
● Proliferation and migration are also stemness properties

Cell Migration
● Microscopy- 2 photon microscope
○ Video registration program in confocal/fluorescent microscope and process all videos/set
of images using ImageJ

Mouse Behavior
● Spatial Learning and Memory
○ Platform in the center of the pool. During training, it must be exposed. During testing,
right below the water. Animal on the platform for twenty seconds. The water maze has 4
positions: north, south, east, or west. Animal swim/search for the platform for a
maximum of 60 seconds. Animals sit on the platform for 15 seconds, Repeat for two
more trials, starting at a different direction for each trial. ACTUAL TEST- make the
water opaque- 3 trials each 60 secs for each starting direction, monitor the path using the
SMART system and videotape each trial.
○ After the 12 trials- one probe trial, in which the platform is removed from the pool. The
probe trial is performed to verify the animal’s understanding of the platform location, and
observe the strategy that the animal follows when it discovers the platform is not there.
Record the number of times the animal crosses the center of the pool during the 30
seconds
● Hyperlocomotion
○ Distance traveled in 10-min intervals for 40-60 min
● Anxiety
○ Social Interaction Test
■ Mean total time engaged in social behaviors is scored- categories of behavior for
each treatment group including aggressive (attack, aggressive unrest), fearful
(vigilant posture, escape and defense activity), social (following, social sniffing,
over-under climbing), and locomotion (rearing, walking during cage
investigation) and report the mean number of events in each category.
 ○ Open field exploration test
■ Mouse in center of chamber- freely explore the chamber for around 5 min. Each
line crossed is scored as one unit of activity. Generally spend more time
exploring the walls than center. Mice that spend significantly more time
exploring the unprotected center area demonstrate anxiety inhibiting behavior.
○ Elevated Maze
● Each placed in the central area of the maze with open access to any arm,
free roam the maze for 5 min. The number of arm entries and the amount
of time spent in the open and closed arms are recorded by an automated
photo beam sensor recording system.
● Mice tend to avoid the open areas, especially when they are brightly lit,
favoring darker, more enclosed spaces
● Extensive t​ ime in one area reflects low levels of exploratory activity
○ Light-Dark Exploration Test
● Mouse is placed centrally into the illuminated compartment allowed to
freely explore for 10 min while transitions and time spent in the dark
compartment are recorded
● Most mice naturally demonstrate a preference for the dark, protected
compartment. The key measure for assessing anxiety-related behavior in
this design is a change in willingness to explore the illuminated,
unprotected area, reflected in increases or decreases in the number of
transitions between the compartments, and in time spent in each
compartment, during a 10-min test session. Treatment with anxiolytic
drugs increased the number of transitions between the two
compartments, without altering the preference of the mice to spend more
time in the dark compartment
● Depression
○ Forced Swim Test
■ In cylinder with water for six minutes with the video recording
■ Floating- depression
○ Tail Suspension Test
■ The animal is hung from a tube by its tail for five minutes- the time until it
remains immobile is measured
■ Immobility- stress/depression
● Fear
○ Fear conditioning
■ Mouse in cage, explore, adverse stimulus, placed in new cage, see if explore or
stay in peripheral
■ Also tests memory

HOW TO CHECK A GENE KNOCKOUT

 ● PCR the mRNA
● Protein analysis with western blot

Between Subjects/Groups Within Subjects

Different people in each condition Same person tests all conditions

REMINDERS
Remember to include tables, predictions, and graphs
Talk about limitations of procedures
General Limitations
● Access
● Expenses
● Time
Implications of the research findings

IV and DV Charts to get you started

Control- wild type Upregulated Downregulated/
knockout

Time
 Criterion 6
Development Articulates clear, reasonable, and succinct research questions and definitions given the
purpose, design, and methods of the proposed study.

All constructs and variables have been appropriately defined.

Research questions are succinctly stated, connected to the research issue, and supported
by the literature.

Application of Procedures are thorough, manageable, coherent, and powerful for generating valid and
research reliable data.
methodologies
Procedures are chronological and replicable.

Detailed descriptions of instruments and observation/protocols are included

Understanding of the A thorough, reasonable discussion of assumptions and limitations is provided.

limitations of
methodology Implications/reflections are insightful and realistic for future research/practice.