Genetic modification Flashcards
Basic vs Clinical research
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
How to decide on which animal to use?
Lifespan
Accessibility of animal
Price
What is a littermate?
one member of a pair or group of animals born in the same mother/litter
Forward vs reverse genetics
Forward: phenotype to genotype (was more common)
Reverse: genotype to phenotype
Steps for forward genetics
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)
examples of forward genetics
Causing spontaneous mutation by introducing a chemical agent (ENU changes a certain base)
Stargazer, reeler
examples of reverse genetics
- 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
Knock out vs in
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
Styles of transgenic models
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)
Zygote injection
Transgenesis by pronucleus injection of naked DNA
- Prepare unfertilised ovum
- Microinject DNA (oocyte卵母細胞) to fertilise the ovum
- Reimplantate the egg for mum’s pregnancy
- Some litter/babies with transgenic traits
KO by homologous recombination in ES cells
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
Essential embryology
- male pronucleus is injected
- zygote
- split split split
- blastocyst complete
- gastrulation
- fetus created
Details on pronucleus injection
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
Example of pronucleus injection
- 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
Issue for pronucleus injection
- 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
Technical requirements for pronucleus injection
- 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
ES cells
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
- Culture ES cells
- Introduce a gene into ES cells (test tube)
- Select cells containing genes into a separate test tube
- Inject transformed ES cells back into blastocyst
- Breed the host mouse
Technical requirements for ESC
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
zygote (pronucleus) vs blastocyst injection
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
Manipulating ESC (Nobel to Mario Capecchi)
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
Construction requirements of a gene replacement vector
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
Steps for constructing gene replacement vector
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
Key for constructing vector for ESC
-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
Steps for gene trap mutagenesis
- Infect ES cells with retrovirus (that has the gene for wanted protein)Instead of retrovirus, gene trapped vector can be used as well
- Screen for X-gal positive colonies
- RT-PCR clones with lacZ primers, sequence, identify interrupted gene
- make mice from gene trapped ES cells (chimeric)
-Remember for gene trap as well, the insertion is random (not targeted)
Key for successful transgenic mice
- Check integration via positive tail biopsy PCR
At which tissue it is expressed - Breed founder with wt mice
- Positive tail biopsy PCR for the offspring
PCR positive = heterozygous F1 off springs - Sacrifice, check tissue of interest for expression
Re-breed correct founder for experiments
No-no for breeding transgenic mice
Always HETEROZYGOUS transgenic founder (e.g. Cre-line) and WILD type; because we do not know the effect of homozygous transgene
CRE/loxP System
-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
Classical point mutation ”Knock In”
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
Inducible point mutation
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
Genetics/breeding schemes
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
Saving money
Make mice Get KO mice/vector from repository E.g. Jackson repository EUCOMM KOMP GenSat EMMA
Targeting Strategy
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
