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M2-5 > Genetic modification > Flashcards

Genetic modification Flashcards

1
Q

Basic vs Clinical research

A 
Basic
Intrinsic function of the gene/protein
Identify location of the certain protein
Outcome of a certain gene knockout/overexpression
Using cell culture (cell line , primary cells  etc)
Translational
Clinical
How the phenotype is like
Treatment for a certain phenotype
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2
Q

How to decide on which animal to use?

A

Lifespan
Accessibility of animal
Price

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3
Q

What is a littermate?

A

one member of a pair or group of animals born in the same mother/litter

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4
Q

Forward vs reverse genetics

A

Forward: phenotype to genotype (was more common)
Reverse: genotype to phenotype

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5
Q

Steps for forward genetics

A

Search for variants or mutants
Increase probability to find mutants through mutagenesis
Crucial: phenotyping
Steps: genotyping/sequencing
Tedious: finding mutated gene/defining mutation
Proof: complementation
(Making sure that disease is caused by the proposed mutation by seeing the outcome of rescuing)

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6
Q

examples of forward genetics

A

Causing spontaneous mutation by introducing a chemical agent (ENU changes a certain base)
Stargazer, reeler

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7
Q

examples of reverse genetics

A
  • Transgenic expression of “disease” gene
  • Targeted disruption “gene knock-out”
  • Conditional knock-out
  • ”knock-in” point mutation
  • ”knowck-down” – KO at after developed stage
  • Reporter/marker
  • ALS
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8
Q

Knock out vs in

A

Knock-outTargeted removal of parts of an endogenous gene “Targeted disruption”; usually in the exon
Knock-inTargeted/non-targeted introduction of mutations into an endogenous gene

Knockdown: KO after developed stage

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9
Q

Styles of transgenic models

A

conventional (e.g. gene trap): when you want to see the global/whole-body effect
tissue-specific: when you want to see effect on a certain cell type
inducible: when you want to see effect later in growth
knock-in:targeted introduction of a transgene into a silent genomic area (commonly targeted: ROSA26)

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10
Q

Zygote injection

A

Transgenesis by pronucleus injection of naked DNA

  1. Prepare unfertilised ovum
  2. Microinject DNA (oocyte卵母細胞) to fertilise the ovum
  3. Reimplantate the egg for mum’s pregnancy
  4. Some litter/babies with transgenic traits
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11
Q

KO by homologous recombination in ES cells

A

Manipulate the germline via embryonic stem cells
1. Prepare ES cells derived by donor blastocyst
2. Inject ES cells to blastocyst
3. Reimplantate the blastocyst to mum
4. mum becomes chimera: a single organism composed of cells with more than one distinct genotype
You want to breed mice with higher ES cell in reproductives (e.g. sperm and egg), but this (as we cannot sacrifice them) can only be estimated by seeing how much of their offsprings are ES rooted
5. Eventually offspring with wanted mutation will be born

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12
Q

Essential embryology

A
  1. male pronucleus is injected
  2. zygote
  3. split split split
  4. blastocyst complete
  5. gastrulation
  6. fetus created
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13
Q

Details on pronucleus injection

A

KEY: Injection of DNA vector (with promoter and gene) in the male pronucleus
Choose a tissue specific promoter to target specific tissue
Promoters e.g. albumin – hepatocytes in liver

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14
Q

Example of pronucleus injection

A
  • Expression of pathogenic genes
  • Expression of negative selection genes
  • Expression of cre-combinase
  • Insertion of a marker
  • Molecular pharming… insertion of genes (in bacteria) that code for useful pharmaceuticals into host animals or plants
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15
Q

Issue for pronucleus injection

A
  • Frequency of insertion is unknown
  • As it is random injection it can harm other gene or gene may land on a wrong location
  • Make sure to have a way to trace back whether the gene is properly introduced
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16
Q

Technical requirements for pronucleus injection

A
  • Fertilised oocytes/zygotes (collected after superovulation with hCG)
  • Designed expression vector
  • Micromanipulators for pronucleus injection
  • Surgical transfer of oocyte back into the mouse
  • Vasectomised stud male
  • Pseudo pregnant foster mothers

-Genotyping/test for transgene expression

17
Q

ES cells

A

ES cells derived from inner cell mass

  • Remain pluripotent and Oct4 positive (marker for pluripotency) in culture
  • Remain to self-renewal into SC when LIF is provided (differentiate if LIF is not present)

Making of mice from ESC

  1. Culture ES cells
  2. Introduce a gene into ES cells (test tube)
  3. Select cells containing genes into a separate test tube
  4. Inject transformed ES cells back into blastocyst
  5. Breed the host mouse
18
Q

Technical requirements for ESC

A

Blastocyst (collected after superovulation with hCG)
Manipulated ESCs
Micromanipulators for blastocyst injection
Surgical transfer

Vasectomised stud male
Pseudo pregnant foster mothers

Genotyping/test for germline transmission

19
Q

zygote (pronucleus) vs blastocyst injection

A

ZYGOTE
Easy vector construction
Relatively fast production of founder animals
Screening of several founder lines necessary
Variable expression in different mouse lines

BLASTOCYST
Sophiticated vector construction
ESC culture required
Slow progress, more generations
Germline transmission is critical
Clear, reproducible results, as lines behave identical
20
Q

Manipulating ESC (Nobel to Mario Capecchi)

A

Construction of a classical targeting vector; vector must have

  • Homologous recombination (over a long range) between genomic and manipulated DNA
  • Positive and negative selection casette

Can be done by
Gene trap
Conditional gene targeting
Multipurpose alleles

21
Q

Construction requirements of a gene replacement vector

A

Must have

  • homologous recombination site
  • positive and negative selction marker
  • Region of homology ideally 5-15kb, homologous arm 1-5kb
    -Positive selection marker: Check whether the recombination occurred
    E.g. often used is neomycin phosphotransferase (Neo cassette) to disrupt the gene
    -Negative selection marker: Check whether the recombination did not include anything extra
    e.g. Herpes simplex thymidine kinase (TK, make sensitive to ganciclovir), Diphteria Toxin A
22
Q

Steps for constructing gene replacement vector

A

Steps
1. Check if proper recombination occurred
To establish targeted ESCs, do electroporation for positive selection (add G418 and only Neo integrant survive) then negative selection (add ganciclovir and illegitimate integrant die)
2. Check if recombination is at right place (no shift frame etc)
Run PCR or southern blotting, Check for expression with qRT-PCR/Western blotting

23
Q

Key for constructing vector for ESC

A

-Make sure to design the right beginning and end
Beginning can have promoter (optional)
End NEEDS poly A tail so the translation stops there
-Grow good ESC cells
ESC grows on a good feeder cell (MEFs from NEO embryos, grown to confluency and mitomycin C treatment)
-LIF
LIF is expensive but essential to prevent cell from differentiating

24
Q

Steps for gene trap mutagenesis

A
  1. Infect ES cells with retrovirus (that has the gene for wanted protein)Instead of retrovirus, gene trapped vector can be used as well
  2. Screen for X-gal positive colonies
  3. RT-PCR clones with lacZ primers, sequence, identify interrupted gene
  4. make mice from gene trapped ES cells (chimeric)

-Remember for gene trap as well, the insertion is random (not targeted)

25
Q

Key for successful transgenic mice

A
  1. Check integration via positive tail biopsy PCR
    At which tissue it is expressed
  2. Breed founder with wt mice
  3. Positive tail biopsy PCR for the offspring
    PCR positive = heterozygous F1 off springs
  4. Sacrifice, check tissue of interest for expression
    Re-breed correct founder for experiments
26
Q

No-no for breeding transgenic mice

A

Always HETEROZYGOUS transgenic founder (e.g. Cre-line) and WILD type; because we do not know the effect of homozygous transgene

27
Q

CRE/loxP System

A

-Cre recombinase mice (Scissor)
require a recognition site (e.g. Lox P) which strategically surrounds the targeted gene
Created by pronucleus injection
-Flox mice
usually made by ESC
LoxP site introduced by P1 phage (bacteriophage) and the homologous recombinase
LoxP can include either target gene or stop codon
-Works by breeding flox mouse (with loxP) and cre-mice for excision (KO) or inversion
-Markers (e.g. lacZ) can be embedded to work as a “reporter” for proper recombination

28
Q

Classical point mutation ”Knock In”

A

Double homologous recombination with mutated gene*
1 Create gene* with lox-neo-lox
Neo function as a positive selective marker
2. Introduce Cre to cut out the neo part
=> gene* is expressed

Cre/LoxP and Flpe/FRT is independent of each other meaning they can be used at the same time

29
Q

Inducible point mutation

A

Later in the development, one can introduce CRE for certain excision
They can also have 2 different loxP sites which requires different recombinase
Application:
brainbow 1.0 (excision)
brainbow 2.1 (inversion)
Inducible KO
E.g. TTR-mER-Cre-mER
Only when tamoxifen is present, Mer-Cre-Mer get exicised
Mer encodes for estrogen receptor that is modified to only react with non-endogenous estrogen
Tamoxifen can be injected/digested; may require few days/weeks for it to accumulate to reactive concentration
May induce undesired physiological side effects

30
Q

Genetics/breeding schemes

A

For global: + wild type - mutated type
e.g. +/+

Heterozygous +/- X +/- is recommended as you can have variation is littermate

For Cre/lox: f for flox w for wild type +w/CRE -w/o CRE
always have one Cre+ and one Cre-
E.g. +f/w X -f/f

31
Q

Saving money

A 
Make mice
Get KO mice/vector from repository 
E.g. 	Jackson repository		
EUCOMM		
KOMP		
GenSat		
EMMA
32
Q

Targeting Strategy

A

KO first allele (promoter-less selection cassette)
The beauty is that within one mice it can create both global KO (w/ Flp later can use cre for inducible KO) and tissue-specific KO (Cre)
Cre sites can also be inducible

M2-5 (4 decks)

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