L6 Genetic tools

Question 1

Forward genetics - gene discovery

Answer 1

see onenote

• Chemical mutagenesis screen
• phenotype => genotype
• random mutagenesis or enhancer trap is screen for an interesting phenotype
• deep sequencing allows for identification of genes implicated
• can be used for novel gene discovery

Question 2

Reverse genetics

Answer 2

Genotype => phenotype

gene knockdown vs knockout

Question 3

Reverse genetics - gene knockdown

Answer 3

see onenote slides

• allows us to study the cellular, organ and behavioural phenotype to assess the function of one particular gene
• used for disease modelling

Question 4

Forward vs reverse genetics

Answer 4

Forward:
- QTL mapping, association mapping, positional cloning, mutagenesis

Reverse:
- genetic engineering, RNAi, TILLING, insertional mutagenesis

Question 5

Forward genetics - fish

Answer 5

see onenote

Question 6

Morpholino (MO) technology

Answer 6

Antisense oligonucleotide

1. 25bp synthetic oligomer complementary to bind mRNA of target gene
2. transcription occured unperturbed, MO acts at mRNA level

two types:

1. translational MO
2. splice MO

MO injected into yolk (or cell) of up to 8 cell stage embryos. At these stages MO can diffuse equally into all cells

can lead to many different changes, may not always be predictable and some protein function may remain

Question 7

MO efficiency

Answer 7

depends on numbers of MO vs number of mRNA molecules

with every cell division, MOs roughly halved and knockdown often becomes inefficient by the end of the rapid developmental period in the first 3/4 days

Question 8

Translational MO

Answer 8

binds start site or just upstream and prevents translation by steric hindrance

mRNA formed but no protein

protein loss can be tested using antibodies against the protein either in immunostaining or western blots

Question 9

Splice MO

Answer 9

binds to donor or acceptor splice site and prevents proper splicing

spliceosome cannot recognise correct site and aberrant splicing leads to a defective protein

Question 10

Splice MO examples

Answer 10

see onenote

should be able to define or label

• exon skipped
• intron
• cryptic intronic
• cryptic exonic

change in mRNA length can be detected by PCR with primers at either end of the relevant region followed by gel electrophoresis

Question 11

MO controls

Answer 11

see onenote

MO controls to address off target/non-specific effects

MO to the wrong sequence/gene or procedural phenotype

1. controls for experimental procedue
2. common sense for phenotype
3. RNA rescue
4. using two different MO at conc that alone show no phenotype - additive effect

Question 12

Maternal effect

Answer 12

see onenote

maternal rescue

Question 13

Conditional and Inducible gene expression

Answer 13

Conditional

• more general
• genes only expressed if a condition is met

Inducible
- specific type of condition which can be controlled allowing us to directly choose when to activate/shut down gene expression

Question 14

Conditional gene expression

Answer 14

see onenote

spatial and temporal control

1. when a specific promoter is activated
   - promoter could be limiting gene expression to a particular tissue/organ/cell type
2. when a specific protein is present (e.g. TF, enzyme)
   - cre/lox system
   - cre recombinase will cut loxP sites - both must be in same cell

Question 15

Inducible gene expression

Answer 15

see onenote

temporal control, can choose when to induce

E.g. induced by temperature or chemical at any time

Hsp70 activated at 37 degrees, can be designed to drive a gene of interest

Cre recombinase - oestrogen receptor fusion (CreER)

• expression conditional with tissue specific promoter
• protein induced when tamoxifen is added (activated by translocation into the nucleus)

Question 16

Zebrabow - conditional and inducible

Answer 16

see onenote slides

in presence of cre recombinase (conditional), different fluorescent proteins are made

construct can be targeted to specific tissues using a tissue-specific “conditional” promoter to confine Cre recombinase

CreERT allows for tamoxifen “inducible” protein function

heatshock promoter driven Cre allows for temp “inducible” transcription

Question 17

Zebrabow uses

Answer 17

1. individual cell tracing

2. clonal analysis - find all the offspring from one progenitor/stem cell

Question 18

Opteogenetics

Answer 18

see onenote slides

A light switch for brain cells
- using optics (lights) combined with genes encoding light sensitive proteins to elicit a change in cell activity e.g. nerve cell firing

two main genes:

1. channelrhodopsin excites neurons
2. halorhodopsin inhibits neurons

allows for direct correlation of one neuron to behaviour, can be possibly used to cure anxiety, addiction, pain etc.

Question 19

In vivo cell and gene imagine

Answer 19

Using reporter proteins to track different cell populations, watch “gene expression” in real time