Gene Regulatory Elements Flashcards
What are some gene regulatory elements?
Enhancers, LCRs and insulators
What are enhancers?
Positive control elements
Long range
Around 200-250 bp themselves
Can be from 1 to >1000 kb from the promotors (gene controlled) and contain multiple transcription factor binding sites
50x more enhancers than promotors
What are LCRs?
Locus control region
Very strong enhancer either 5’ or 3’ of the gene
Discovered as essential elements to generate transgenic mice
Often found as strong DNase I hypersensitive sites
Bound by many tissue-specific transcription activators
Gives position-independent, copy number-dependent expression
Promotor + LCR will take place - promotor + enhancer is not strong enough to take place
The copy number dependent expression of the transgene
What are insulators?
Prevent interference by regulatory elements in neighbouring genes
Insulators block enhancers from interacting with the wrong promoter
Long range
Transcription Factors that bind to the insulator prevent the enhancer from contacting the promoter
What is another function of insulators?
They block the spreading of heterochromatin
This “barrier” function is also mediated by transcription factors binding to the enhancer
This function is important to “protect” genes from silencing
What assays can be used to see the regulation of gene expression?
Reporter constructs
Use 2 plasmids - one with and one without the enhancer to see the effect on expression
The transfection needs to be even so a crucial control = co-transfect second plasmid, e.g. that expresses a different luciferase to normalise between transfections
Can be into cell lines of type where gene is normally expressed
Can be into heterologous cell lines together with co-transfection of an expression vector for the transcription factor(s) that bind a particular site
How can we predict enhancer sites?
Bioinformatics
Use ChIP seq data - and map the data to the genome (align them)
Typical features of enhancers: DNase I hypersensitive ATAC sensitive H3K4me1 p300 binding Med1 binding Binding of tissue-specific transcription factors
Describe evidence for enhancers blocking insulators?
Used stable transfection of reporter constructs
The insulator has enhancer blocking activity when in between the LCR and promotor
But not when flanking them
Expression of neo conferred resistance to G418 – colony assay
Control – use pieces of lambda bacteriophage DNA – no enhancer blocking activity
Describe evidence for insulators blocking the spread of heterochromatin?
Used stable transfection of reporter constructs
Two insulator elements were used to flank an IL2R reporter construct
(IL2 receptor is expressed on the cell surface and expression can be determined by FACS)
In the absence of an insulator, the reporter gene became repressed, followed by heterochromatin formation
Between 40 and 100 days, most stable cell lines where the construct lacked the insulator, lost expression of the reporter gene – shift to the left of FACS plot
In the presence of the insulator, expression and euchromatin was retained
Give an overview of the generation of transgenic mice?
Transgene = Gain of function Foreign DNA is stably introduced The transgene (linear DNA) is injected into the male pronucleus of one cell embryos Foster mothers are made pseudopregnant by mating with a vasectomised male
Insertion of the transgene into the genome is random
So when they are born - saliva is taken and PCR to see if the transgene has been inserted
Make transgenic founder mice - let these breed to ensure the transgenic gene is present in all cells in all generations
Transgenes insert in head-to-tail manner
What are used to generate transgenic mice?
Vectors
In all cases the DNA is isolated from the vector backbone prior to injection of the transgene
Plasmid vectors – insert up to 20 kb
Cosmid Vectors – insert up to 45 kb
BAC Vectors – inserts 100-300 kb
YAC vectors – inserts up to 1 mb
What are the advantages of analysing gene expression via transgenic mice?
In vivo; transgene is exposed to factors at correct stage of development
Reporter genes can be used to analyse when promoters are activated during development
What are the disadvantages of analysing gene expression via transgenic mice?
Copy number is different from line to line
Therefore you need to count these and account for the different copies when analysing your data
Insertion site is random
I.e. In heterochromatin, or insertional mutagenesis (in an active gene)
Safe harbor sites (SHS) are genomic loci where genes or other genetic elements can be safely inserted and expressed - as they are always in euchromatin
Use of LCRs and insulators can help to overcome position effects
Need to analyse many transgenic lines to ensure that the effects are real
Give an overview of the generation of knock-out mice?
Knock-out = Loss of function
- Isogenic transgene SNA is introduced into embryonic stem cells (pluripotent) - e.g. By electroporation
- Drug selection is used and the surviving colonies are screened for the transgene
- Characterised targeted cells are microinjected into 3.5 day mouse blastocysts
- Blastocysts are transferred into a 2.5 days pseudopregnant recipient mouse (gestation for 21 days)
- Chimaeric offspring are identified and mated to term for germline transmission of the transgene
What are the different methods of targeting the vector for the generation of knock-out mice?
Gene targeting
There are regions of homology - that must perfectly match for homologous recombination
The flanking region should be the same, just with the addition of our mutation
Random integration
This takes place more often
Select to thymidine kinase - and kill them with gancyclovir - as these ones will have been randomly placed (unwanted)
Give the mechanism for generation of knock-out mice - step 1 to 3?
Step 1
Isolate a developing embryo at blastocyst stage e.g. from a mouse with grey fur
Grow stem cells in tissue culture
Step 2
Transfect stem cells with homologous recombination construct
Positively select with neomycin and negatively select with gancyclovir
To remove random integration but select for neomycin resistant
Check for recombination by Southern blotting or PCR
Step 3
Remove homologously recombined stem cells and inject into a blastocyst that would generate a mouse with white fur
Implant several chimeric blastocysts into a pseudo-pregnant mouse with white fur
Give the mechanism for generation of knock-out mice - step 4 to 6?
Step 4
The mother will give birth to a range of mice
Some will have normal white fur
Others will be chimeric mice that have some cells from the white fur blastocyst and some cells from the recombinant grey fur ES cells
Step 5
Mate the chimeric mice with wild type white fur mice
If the germ cells (oocytes/sperm) of the chimeric mice were derived from recombinant stem cells, all the offspring will have grey fur
In this case, each cell in these mice will be heterozygous for the knock-out
Step 6
Mate the heterozygous grey mice (+/H) and genotype the offspring via PCR or Southern blotting
The homozygous recombinants (H/H) are the knock-out mice that are bred as the knock-out line
What are the disadvantages of knock-out mice?
15% of knock-outs are embryonic lethal
Some knock-outs show no effect – compensation by closely related gene product
If the gene works in combination with other genes, the effects are not always fully apparent in all organs
What are conditional knock-outs?
Inactivate a gene only in specific tissues and at certain times during development
Cre-Lox technology
Cre = a site-specific recombinase from phage P1
This recognises a 34 bp DNA sequence – loxP - and cleaves it
Flank gene of interest with 34 bp loxP sites (floxed)
If Cre recombinase is expressed, gene between the loxP sites is removed
Describe the generation of conditional knock-out mice?
This takes place by flanking with LoxP sites
Cre Recombinase causes recombination between these sites and thus deletion of the intervening DNA
Cre mice express recombinase in specific cells
Generate chimeras
Breed mice with targeted allele
Describe Cre recombinase?
Cre recombinase can be used to inactivate or activate gene expression
Cre recombinase can inducibly cause changes in gene expression
Tissue-specific expression of Cre causes changes in the target gene only in those tissues
Inducible Cre allows target gene inactivation at a controlled time
How is Flp used with Cre recombinase?
Neomycin can cause local chromatin repression
So now we flank the neomycin resistant gene with Flp recombinase
This allows removal of selection marker prior to generation of conditional knock-out mice
Select the ones that are no longer neomycin resistant
Describe the placement of the LoxP sites?
This allows a number of different conditional rearrangements
- Excision - cis placement of loxP sites in the same directional orientation
- Inversion - cis placement of loxP sites in opposite directional orientation
- Translocation - trans placement of loxP sites
What is CRISPR-Cas9?
This technology has revolutionised the generation of K/O mice
Cleaves DNA specifically 3 nt away from the PAM sequence
Relies only on Cas9 and a single guide RNA
The guide RNA can be determined by the experimenter
What is the timeline of knock-out mice with CRISPR?
Knock-out mice can now be generated in 6 weeks
Mating -> zygote = 20 hours Zygote -> Zygote injection = 3 hours Embryo culture = 24-76 hours Embryo transfer = 0.5-2.5 days Genotyping at 3 weeks after birth
What can CRISPR generate?
Multiple different genome changes are possible – with very high efficiency
Possible to generate: Single point mutations Gene tags Gene deletions Conditional (Floxed) alleles Insertion of marker genes