Genetic Manipulation Technology 3&4 Flashcards
Define transcription
Transcription is the process of turning DNA into mRNA by RNA polymerase II
What two components are required by RNA polymerase to start transcription
Enhancers
Core promotors
Define enhancers
Enhancers required to increase RNA polymerase II activity at the promoter.
Some cells require more than one enhancer
Give some characteristics of enhancers
Variable distances from the start of the gene
Can work on more than one gene
Dont produce protein product
Indicate when, where and how much of a gene is sequenced.
What is the use of enhancers in genetic cloning
Can be isolated and placed next to the gene in genetic cloning
Define promotors
Promoters are DNA sequence immediately next to transcriptional start site of the gene coding region (exons).
Found at the pre-initiation complex (PIC) - RNA polymerase etc
Can promoters transcribe on their own
Usually not, need enhancers to bring DNA binding transactivators
Once the transcribed mRNA is formed, what is left
An mRNA sequence with introns and exons (coding regions)
What happens to this mRNA sequence before translation
Capping at 5” end to stop degredation
Poly A tail at 3” end
splicing of introns - different splicing combos make different genes
Define translation
The process of changing a spliced mRNA sequence into protein
How does translation work
spliced RNA enters ribosome and is paired with tRNA equivalent which starts and continues a chain of amino acids
What is a reading frame
A stretch of successively arranged codons
What is an open reading frame
a reading frame without a Termination Codon in more that 50 codons
Define a transgenic animal
An animal which can stably contain an exogenous gene that has been artificially inserted.
These genes can usually be inherited.
All cells contain this gene (inserted embryonically)
How to build a transgene - 5 stages
Isolate the gene and form cDNA (gene with no introns) - easier to handle as smaller
Ensure promotor sequence before gene and regulatory sequences (transcription factors)
Ensure correct RNA processing - ensures poly A tail
Genetically ligate an intron as mRNA processing works better with an intron present
Termination sequence
Why is it difficult to get transgenes into cells
Transgenes are hydrophilic while cell membranes are hydrophobic
Give 4 ways how transgenes enter cells
Direct micropipette injection
Electroporation - shocks cells to make a hole to let DNA in
Chemical transfection - Incubate cells in a culture medium containing DNA and a chemical that wraps the DNA up diffuses through the cell membrane meaning the transgene can enter the original DNA
Infection - expose cells to viruses carrying transgene DNA that will infect cells
How to make all cells of an animal have the transgene
Enter into one cell embryo
What happens if the transgene is added to a multicell embryo
Mosaic mouse (some cells have it some not)
What is the most efficient way to enter a transgene into a one-cell zygote
Direct injection with a micropipette then insert back into animal
What are some issues with direct injection
Relies on the DNA replair enzymes integrating the DNA
Often the transgene will integrate as a concatemer (multiple copies of the same gene in a row) - we have no control of how much is copied.
Give some issues of transgenic animals
Weak promoter
Excess copy number
Position of integration into genome - can get expressed in the wrong organ
Epigenetic modification - if repeat is too big it is shut down usually
Genetic background of animal used as the subject
Not all the subjects will be expressing then gene or even carrying it
What type of transgenes work best
Very big transgenes where the promoter is right at the start usually work best
What is viral-mediated gene transfer
Using viruses to get transgenes into cells
How do retroviruses work
Infect cells - RNA gene is reverse transcribed into DNA and enters host genome
Virus DNA contains all the genes for proteins needed to make a more infective virus
What is the gag gene of a retrovirus
Encodes proteins of nucleprotein core of viron
What is the pol gene of a retrovirus
Encodes reverse transcriptase and integrase
What is the env gene
Encodes surface proteins
What is a packaging cell
A cell which has the gag, pol and env genes to make the viral particles
What is the purpose of packaging cells
Allows the transgene to enter with a packaging signal and all the other viral proteins are formed –> make viral subunits with the transgene in them and burst out the cell –> this is collected and used to infect other cells
Why are viruses not usually used to create transgenic animals where the gene is found in every cell
Embryos are usually able to spot and silence viral sequences by methylating them
When are viruses used in transgenic animals
Usually adults or mid-gestation embryos therefore creates a mosaic
What 3 other viruses types are used
Lentiviruses (retroviruses type)
Adeno-associated virus AAV
Adenoviruses
Features of lentiviruses
HIV derived and very efficient and stable for long term expression of transgene
Features of adenoviruses
DNA genomes that are large
Short-term (few weeks until killed by subjects immune system)
Dont integrate
Features of adeno-associated viruses AAV
Much smaller than AV
Can infect non-dividing cells and can integrate so popular for gene therapy
Why are viruses good for transgenes and name 2 downsides
Can infect all organisms but may only work on dividing cells and can also be silenced
Can mutate and spread
Why are conditional gene/tissue specific knockouts done
Sometimes when genes are knocked out the organism doesnt survive long enough to determine the true function of that gene. Therefore the organism must be left to reach gestation and the gene must be knocked out there after
Difference between homologous recombination and tissue specific knockout
Homologous recombination - knockout of all genes as embyro
Tissue-specific knockout - after gestation
What is cre-lox technology
Cre is a site speficic recombinase enzyme because it recognises LoxP (34 base pair DNA sequence)
Used in vivo to integrate bacteria DNA
How is cre-lox technology used in kockouts
Between two LoxP sites the cre removes that genetic sequence
What is a floxed gene
A gene that has loxP sites either side so cre can remove it
How is a tissue-specific knockout carried out
One mouse with a floxed gene of interest (LoxP either side)
One mouse with a cre recombinase enzyme in tissue-specific site (e.g. pancreas)
Allow them to breed –> gene is knocked out in ONLY the tissues where cre is expressed
How is a floxed gene formed?
Homologous recombination in embryonic stem cells (using targeting vector and abx resistance marker)
Positive negative selection takes place and sequence surviving cells to make sure only floxed genes are found.
Inject floxed genes into blastocysts then get rid of abx resistance gene as can interfere with floxed gene.
How is the neomycin resistance (Abx resistance marker) gene removed
Breed the mice with transgene and the abx resistance marker will be knocked out due to the floxed site being found before neomycin site
-> will now get flexed allele
What is difficult about using the cre-loxP construct
Finding a robust promoter which is strongly pro-cre expression only- it is the rate limiting step
How would you control when a gene is knocked out in a subjects lifetime
The expression of cre can be controlled in a timely matter in 2 ways -
Tamoxifen
Tetracycline
How does tetracycline cause cre knock in at controlled time
In normal rat…
-The Tet-activator protein ⟶ Binds to the tet operon (tetO) to activate transcription of Cre
-BUT not in the presence of tetracycline (as this abx will bind to tetA)
SOOOOO……..
-if you keep mice dosed with tetracycline
-> the tetracycline binds to tet activator protein (to stop it working.)
When tetracycline removed…
-> TetA binds tetO
-> Cre is transcribed
-> floxed gene knocked out
summary:
If keep mice dosed with tetracycline - binds to tet activator –> The cre gene isnt turned on.
so the mice survives until the mice isnt given tetracycline anymore.
- allows the cre gene to be activated and knocked in replacing the floxed gene
tet-off system:
tet A protein bend to tet O to activate cre transcription when no tetracycline, tetracycline blocks this
How does tamoxifen induce cre expression at a controlled time
Cre-ER (Cre with an eostrogen receptor) is used to allow it to bind to tamoxifen, when this occurs, cre-mediated recombination is carried out
Features of Cre LoxP
Established
Not 100% efficient
Not reversible
Features of tetracycline/tamoxifen inducible systems
Sometimes routine
Efficient
Reversible
Why are adenoviruses good for gene transfer
EXTRA READING - LEE
Easiest viral transfer - most humans have an adenovirus receptor and integrin receptor meaning the virus can easily invade humans and produce high levels of transgene.
Gutless adnoviruses - less of an immune response by humans so last longer to make transgene
A lot of research now has established safe doses and routes
Doesnt integrate into host genome so stops any mutagenesis therefore increasing successful expression of transgene
Give an example of adenoviruses being used to treat conditions
1992 - Alpha-1 antitrypsin given to a person with alpha-1-antitrypsin deficiency via adenovirus into the person hepatocytes
How have adenoviruses been used as anticancer agents (gendicine)
EXTRA READING - LEE
Gendicine is a recombinant adenovirus engineered to express wildtype-p53 (rAd-p53) in tumour cells. This virus is designed to treat patients with tumors which have mutated p53 genes.
It also blocks VEGF in tumour cells preventing it from growing
First ever gene therapy
What is rosa26 good for
Hoinstein et al. 2008
Prevents transgene instability and therefore epigenic modification through methylation etc.
Over 130 knock-in lines have been created based on the ROSA26 locus.
A human homolog has been identified of the ROSA26 locus - a site that can be used for genetic therapy in humans.
Why are viruses poor choices for making a fully transgenic animal
Because of long-term silencing of viral gene in eukaryotic cells
How does Forni at al. 2006 give an example of tamoxifen-induced cre-loxP knockout
Uses cre-loxP tamoxifen to show how high levels of cre cause hydrocephalus in mice.
The cre mice and loxP mice were allowed to mate to show how cre causes microcephaly and hydrocephalus by having low levels of neural progenitor cell proliferation and high levels of cell death.
However, the CreER animals were left unaffected when tamoxifen was added later in life. Showing that Cre is damaging to neural progenitor cells before birth and that some types of hydrocephalus are due to defects in neural precursor proliferation.
What issues did Hofman et al. run into in 2006 regarding transgenic animals
1/3 of those pigs studied with a lentiviral injection of transgenes experienced epigenetic modifications and were methylated to silence them.
What does Simmons 2013 tell us about hybrid dysgenesis
P elements are transposable elements meaning whole sections of DNA can be moved elsewhere autonomously.
The P element codes its own transposase to allow it to be cleaved and repositioned elsewhere. They are found in humans and can be used for enhancer trapping - inserts P element near enhancer to study activity of the enhancer and the genes associated with it
What does Nicholas et al state regarding HIV thymidine kinase in 2003
The herpesvirus thymidine kinase gene has also been used as a “suicide gene” as a safety system in gene therapy experiments, allowing cells expressing the gene to be killed using ganciclovir. This is desirable in case the recombinant gene causes a mutation leading to uncontrolled cell growth (insertional mutagenesis).
Has been used in animals to kill of tumour cells
How can inverted loxP sites be used in condition cell ablation
Extra reading - Kmita and Gregoire 2008
Previously shown that loss of the loxP carrying chromosome can occur when loxP sites are arranged in inverse orientation. By adding cre-mediated recombination it not only causes chromosomal loss but also triggers apoptosis.
Targeted recombination between inverted loxP (TRIP) triggers cell death in proliferating cre-expressing cells and can therefore be used to ablate specific tissue types where cre is expressed
Give an example where inverted loxP sites are being used to show cell apoptosis
Crossed mice having a set of loxP sites in inverted orientation
within the HoxD gene cluster with mice carrying a Cre transgene expressed primarily in developing limbs We found that limb buds of invloxP/; Prx1-Cre embryos were severely reduced in size as compared with wild-type ones.
In contrast, embryos carrying only the invloxP allele OR the Prx1-Cre
transgene were indistinguishable from wild-type embryos and were used as controls in subsequent analyses.
13,000 cre-positve GFP-positive cells remained per mutant limb bud,
whereas control buds contained 470,000 GFP-positive cells
This suggests cell death occurred as a result of chromosome elimination
What is the role of exon5a in PAX6 gene
WIDER READING - Signh et al 2002
Found out role of exon 5a was by knocking only this section out of Pax6 gene - needed to make iris.
How do we target cre-recombinase to the tissue of interest?
Drive Cre from the promoter of a gene that
Is only expressed in the tissue you want (quite rare) OR
Is expressed in areas that overlap with your tissue of interest
OR
Create a ‘Designer’ Promoter…
Make Transgenic Mouse carrying Cre on your Promoter, and Check Expression
(If you are making a Cre trans gene, by the injection of DNA into a nucleus of a cell, what is the rate limiting step?) already covered more fancy words
the isolation and cloning of a reliable and robust promoter construct that drives strong Cre expression only in the places where you want it, has been the rate-limiting step for production of tissue-specific knockouts
What is tamoxifen induction of cre expression?
fusion protein combining activity of cre and a mutant form of ligand binding domain of oestrogen receptor
does cre-ER bind oestrogen?
no
what does cre-ER bind?
4-OH-tamoxifen
what happens to cre-ER if no tamoxifen?
Hsp90 grabs cre-ER and keeps it in cytoplasm so no recombination in nucleus
what happens to cre-Er if tamoxifen present?
cre-TRM released from Hsp90 and recombination
