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)
