Gene-Modifying Methods in Neurobiology

Knock Out (KO)

Knock Out (KO)

Ablation of a gene within the genome

Knock In (KI)

Insertion of a gene at a targeted location in the genome (usually used to insert a mutation or a reporter gene)

Knock Down

Reduction of a gene expression (through siRNA (silencing RNA) inserted mutation, expression of Dominant-Negative forms or modification of promoter sequences)

What are some Knock Down Methods

siRNA inserted mutation

Expression of Dominant-Negative forms

Modification of promoter sequence

Transgenic

Random insertion of a foreign gene (from another species)

Is the location in the genome of a transgenic gene modification controlled?

No

Coding Sequences

Sequences that record information for protein synthesis

Fluorescent Proteins

Proteins that emit light upon excitation, used for visualization

Dominant-Negative Proteins

Modified proteins that interfere with the function of the wild-type protein

Cannot sustain the same function as the endogenous

Beta-Galactosidase (LacZ)

An enzyme used as a reporter that reveals its presence through colorimetric assays

Colorimetric Assays

Analytical technique that detects the presence or concentration of a substance by measuring the change in color that results from a chemical reaction

Exogenous Proteins

Proteins that are not normally present in the cell, introduced from outside

Exogenous proteins allow…

Registration of the neuronal activity

Modification of neuronal activity

Induction of neuronal death

Tracing of synaptic connections

GCamP

Fluorescent Ca2+ sensor

Fluorescence in the presence of high concentrations of Ca2+

Optogenetics

A technique to control neuronal activity using light

DREADD

Designer Receptor Exclusively Activated by Designer Drug

DREADD

Used to modify neuronal activity pharmacologically

Provides a ligand to modify the activity

Either increases or decreases the activity of the neuron

DTA toxin

A toxin used to induce neuronal death

System for co-expression via Fusion protein

Fusion of the protein of interest and a Tag (ex: GFP, HA)

Multiple proteins simultaneously expressed in same cells at same temporal pattern

We know that since they are fused, when one is present, the other is too

What process is displayed?

System for co-expression : fusion of protein of interest (POI) and a tag

Limitation of co-expression via Fusion protein/Tag

Shape/space (3-D organization) is altered

Fused might be bigger and not addressed properly in the cell

Coexpression with a fusion protein starts with one single mRNA and results in ___.

One single protein

IRES

Internal Ribosome Entry Site

Coexpression of a protein of interest and reporter through IRES starts with one single mRNA and results in ___

2 independent proteins

What process is displayed?

Coexpression via IRES

The location (inside cell) of the reporter protein is ___ of that of the POI.

Independent

→Both are within the same cell, but precise sub-cellular location of both might differ

Limitation of IRES

Level of expression of POI is higher than reporter protein (imbalance)

Advantage of IRES over Fusion Protein

Since the genes are not fused, there are no potential issues related to altered protein function or conformational change

Advantage of Fusion Protein over IRES

Often expressed more efficiently because their entire construct is treated as a single gene, and translation of both proteins is driven by a single promoter

→Eliminates the need for 2 independent translation events

RNAi

Interference RNA, a method to inhibit target gene expression by degrading mRNA

siRNA

short interfering RNA

2 Strands of siRNA

Guide and Passenger strand

siRNA Guide Strand

Directs silencing - binds to complementary mRNA molecule

siRNA Passenger Strand

Degraded during RNAi process

shRNA

short hairpin RNA

Sequence Carrier

Can be a plasmid or virus

Determined by the length of the sequence of interest to be expressed

Determines the entry path and the duration of the expression in the cell

Which carrier type will be used if the sequence is long?

Plasmid

Why plasmids for long sequences?

Limited space in the genome of a virus

Plasmid stays in cytoplasm → cell will eventually get rid of it

Transfection

Entry of one or several plasmids into cells using chemical compounds to make the cell permeable

RNAi Principle

Induces the degradation of the target mRNA and thereby blocks the synthesis of the protein

→No more protein = No more protein expression

Gene Gun Method

Entry of plasmids by bombarding cells with particles coated in plasmids

Electroporation

Entry of plasmids into cells through the application of an electric shock

Viral Transduction

Entry of genes into cells via viruses, which can integrate into the host genome

Spatial Specificity

Control over which cell type expresses a gene by choosing appropriate promoters

Temporal Specificity

Control over the timing of gene expression during development

Inducible Promoter

A promoter whose activity can be experimentally triggered at a specific time

Recombination

Change the arrangement of DNA inside the genome using enzymes

Cre-recombinase

An enzyme that recognizes LoxP sites for targeted recombination

Flippase-recombinase

An enzyme that recognizes FRT sites for targeted recombination

CrispR/Cas9

A genome editing technique that makes a cut at a targeted site in the genome

Guide RNA

RNA that directs the Cas9 protein to the specific site in the genome

Homology Directed Repair

A repair process that replaces the endogenous sequence with a donor template

Donor Template

A homologous sequence used to control what is inserted during genome editing

Reporter Proteins

Lab-engineered genes that light up (report) when they bind to a specific chemical (like a neurotransmitter).

Entry Path

Route for genetic material into cells.

Carrier

Plasmid or virus used for gene delivery.

Transitory Expression

Temporary gene expression in cells.

Permanent Expression

Long-lasting gene expression via integration.

Plasmid

Circular DNA that remains in cytoplasm.

Virus

Pathogen that can integrate into host genome.

Transfection

Introduction of one or several plasmids after application of chemical compounds that make the cells permeable (in vitro)

What are the methods of transfection?

Electroporation

Transfection

Gene Fun

Gene Gun Method

Plasmid delivery via particle (coated in plasmids) bombardment.

Gene Gun Uses

in vitro slice for nervous system

Injection of fluorescent dyes

Electroporation

Electric shock creates pores for plasmid entry.

What is this image displaying?

Transfection

What is this image displaying?

RNAi

What is this image displaying?

Gene Gun Method

What is this image displaying?

Electroporation

In Vitro

Experiments conducted in controlled environments outside living organisms.

In Vivo

Experiments performed within a living organism.

Calcium Phosphate

Chemical used to facilitate plasmid entry.

Liposomes

Lipid-based carriers for plasmid delivery.

Transduction

Gene delivery using viral vectors.

Cell Tropism

Virus's preference for infecting specific cell types.

Lentivirus

Virus that integrates into host genome. Integrated genetic material for long-term gene expression

Adenovirus

Virus that does not integrate into host genome.

Can infect a wide range of cells, but do not integrate into genome → transient expression

AAV (Adeno-Associated Virus)

Rarely integrates into genome (very restrictive in transgene size they can carry)

GFP

Green fluorescent protein used as a marker.

Trans-synaptic Propagation

Virus spreads across synapses in nervous system.

Soft-skill Phase

Period for postnatal needle applications.

Spatial Specificity

Determines which cell type expresses a gene

Chooses the promoter that allows expression

Ubiquitous Promoters

Promoters expressed in all cell types.
Not Specific
Allows strong expression levels of sequences
Ex: CMV, PGK, SV40, CAGG

Neuron-Specific Promoters

Promoters active only in neuronal cells.
Even if it gets into a different cell, it won't be expressed
Ex: Β-Tubulin, CamKlla, NSE

Neuronal Subtype Specific Promoters

Promoters for specific neuronal subtypes.
More Specific
Ex: TH, VGluT, VGAT

What is an inconvenience of more specific promoters?

Trigger lower expression levels compared to ubiquitous

Temporal Specificity

Expression control at specific maturation stages.
Ex: Nestin, DCX

Inducible Promoter

Promoter activated experimentally at a chosen time.
Most common
Promoter usually off, but you can trigger it when you want

Examples of Inducible Promoters

Tet System (promoter TRE); HSP System; Operon Lactose System (IPTG)

Tet Inducible System

Tetracycline-controlled transcriptional activation system.

The Tet System controls ___ specificity

Temporal

tTA

Tet TransActivator (Tet-OFF)

Spontaneously Active

→Contains tetracycline repressor (TetR) and transcriptional activation domain

rtTA

Reverse Tet TransActivator (Tet-ON)

Spontaneously inactive

tTA is active in the ___ of Doxycycline (Dox).

Absence

rtTA is active in the ___ of Doxycycline (Dox).

Presence

In the Tet-OFF (tTA) system, the presence of Dox causes the ___ of gene expression.

Repression

In the Tet-ON (rtTA) system, the presence of Dox causes the ___ of gene expression.

Activation

tTA has a(n) ___ relationship with doxycycline.

inverse (+Dox → NO expression)

rtTA has a(n) ___ relationship with doxycycline.

direct/proportional (+Dox → Expression)