CMB2000 Flashcards
stages of cloning
- bioinformatics searching
- design primers to include 2 restriction sites
- PCR
- create clean insert with appropriate ends
- plasmid choice - treat with same restriction enzymes
- ligase - to join inset and plasmid in MCS
- transfer/transform into impotent E.coli
- grown on selective media
- pick right colonies PCR straight from colony/culture/plasmid
- mini-prep
what is PCR
- polymerase chain reaction
- amplification of DNA
stages of PCR
- starts with a single stranded piece of DNA
- uses taq polymerase for repeated cycles
- with each cycle there is an exponential increase in strands
what 2 things are needed for replication in eukaryotic cells
- template DNA
- polymerases
why PCR
- sensitive - can amplify as little as one molecule of DNA
- specific - can amplify a unique target sequence stringency depends on temperature and [mg2+]
- cheap
- rapid - results available in a few hours
- robust - DNA is very stable can be amplified from old and degraded samples
what’s in the tube
- template - double stranded DNA
- 2 primers - to prime synthesis
- polymerases - copies the template, extending from the 3’ end of primer
- dNTPs - deoxyribonucleotide triphosphate
- magnesium - co-factor for DNA polymerase enzymes
- buffer - maintain pH and provide necessary salt
what are the 3 regions of tax polymerase
- synthesis
- proof reading
- primer removal
what are the key parts of primer design
- 2 primers - one for each strand
- length 18-24 bases
- 40-60% G/C content
- start and end with 1-2 G/C pairs
-melting temperature of 50-60 degrees - 3’ end must be complementary to the template DNA
- primer pairs should not have complementary region
importance of magnesium
- co-factor
- a non-protein component of the reaction that’s needed to enable the activity of the catalysis
- magnesium acts to enhance the enzymatic activity thereby supporting DNA application
what is the buffer used
- optimal pH is 8-9.5
- tri Hcl
- potassium ions - promote annealing (may be replaced by ammonium sulphate, which destabilises base pairing bonds
names of the 3 stages in PCR
- denaturation
- annealing
- elongation
what is the process of DNA synthesis
- 1st cycle - synthesis of a strand of DNA in test tube
- 2nd cycle - synthesis of two strands in a test tube
- the rest - simultaneous synthesis of both strands
- polymerase chain reaction
- exponential amplification of DNA polymerase chain reaction
how do we detect PCR production
- run products on agarose gel
- use intercalating dye to stain DNA to determine size and yields
what can be identified by detection of PCR
- molecular weight markers
- PCR products
- primers
- template
why is PCR so important
- good when DNA is scarce
- manipulate DNA
- detection of pathogens
- diagnosis of genetic disease
- detecting genetically modified material
- biotechnology
examples of use of DNA
- forensic analysis of DNA samples
- manipulate DNA - genetic modification
- knock out genes - study gene function
- fuse host proteins with GFP
what are the stages of reverse transcriptase PCR
- convert RNA to cDNA. use reverse transcriptase, retroviral enzymes that converts RNA to DNA
- amplify DNA by PCR
what are the sources of RNA
- gene expression (mRNA) - disease vs healthy/drug effects/environment changes
- RNA virus infection levels
what are the ingredients in RTPCR
- template (RNA)
- primer
- dNTPs
- reverse transcriptase
- buffer
features of endpoint PCR
- cheap
- semi-quantitative at best - band intensity
- sequence, genotyping, cloning
- see results at end, plateau
features of real time PCR
- more expensive
- quantity of PCR is proportional to amount of template
- quantification of gene expression, microarray verification, quality control and assay validation, SNP genotyping, copy number variation, viral quantification, siRNA/RNAi experiments
- measures at exponential phase - more precise
how do we include fluorescence
- SYBR gree
- binds to groove of dsDNA –> increases fluorescence
why do we need reference genes
- constant level of expression - not affected by experimental factors
- essential to support validity of qPCR results
- confirms RNA extraction was good and efficient
- supports conclusion of expression levels
examples of reference genes
- beta actin
- GAPDH
- albumin
- 18s rRNA
- TATA sequencing binding protein
what’s in a PCR reaction tube
- DNA template - the sequence to be amplified
- primer (reverse and forward) - to start the synthesis of the new DNA strand
- nucleotides (dATP, dCTP, dGTP, dTTP) - building blocks for the new DNA strand
- taq polymerase - to catalyse the synthesis of the new DNA strand
- buffer - to maintain optimal pH for synthesis
- mgcl2 - essential for tax activity. concentrations of mg2+ ion effects stringency of primer binding
what are the common uses of PCR
- genotyping the patient
- genotyping the pathogen
- phenotyping the disease
what can genotyping the patient be used for
- diagnosis of genetic traits
- detection of carrier of genetic traits
- tissue matching
- predicting the response to drugs (pharmacogenetics
what is HLA typing
- the proteins encoded by HLAs are those on the outer part of the body cells that are unique to that person
- any cell displaying that persons HLA type belongs to that person and therefore is not an invader
what can genotyping the pathogen be used for
- diagnosis of species and strain of infecting pathogen
what can phenotyping the disease be used for
- measuring disease progression
- measuring disease severity
what are the 2 PCR techniques used in genotyping the patient
- PCR-RFLP - restriction fragment polymorphism
- ARMS-PCR - amplification refractory mutation system
what is an allele
any of the alternative forms of a gene that may occur at a given locus
what is a restriction enzyme
an enzyme that digests DNA at a highly specific site
steps of PCR-RFLP
- identifies allelic variants based on presence/absence of a restriction site
- amplify
- cut PCR product with restriction enzyme R
- size-fractionate by gel electrophoresis
what can be identified by restriction site (cutting PCR product)
- if the RE site in neither allele - homozygous allele 1
- if the RE site in both alleles - homozygous allele 2
- if RE site in one allele - heterozygous
what can be identified by restriction site (gel electrophoresis)
- if RE site in both products - homozygous for the disease allele
- if RE site in neither products - homozygous for healthy allele
- if RE site in one of the products - heterozygous
what is sorsbys fungus dystrophy
- example of genotyping the patient
- degenerative eye disease leading to blindness
- mutation in TIMP3 gene introduces premature stop codon
what are the advantages of PCR-RFLP
- cheap
- easy design
- applied to microindels and SNPs
- simple resources
- commonly used technique
what are the disadvantages of PCR-RFLP
- only possible if the site contains a known RE site
- some RE are expensive
- only possible if a single nucleotide variation
- hands on and time consuming
- not suitable for high-throughput
importance of ARMS-PCR
- detects allelic variants using allele specific primers
- simple method for detecting any mutation involving single base changes or small detections
- presence or absence of a PCR product is diagnostic for the presence or absence of the target allele
how can ARMS-PCR be used to diagnose cystic fibrosis
- mutations of the CFTR gene leads to imbalances of cl- transport across plasma membrane
- f508 mutation is the most common cause
RFLP VS ARMS (RFLP)
- uses locus specific primers (will amplify all variants of the chosen DNA sequence
- relies on the presence or absence of a restriction site to distinguish between variants
RFLP VS ARMS (ARMS)
- uses allele specific primers
- relies on the stringency of the PCR to distinguish between alleles
- alternative is tetra primer ARMS-PCR, which uses addition non-allele specific primers
what can genotyping the pathogen be used for
- identifying the species and strain of an infectious pathogen by isolating a specific gene/piece of DNA
- this information will influence: patient management and infection control measures
PCR vs conventional microbial diagnosis (PCR)
- sensitive - can detect single copy of genome
- specific - can identify species and strain
- sensitivity means no need for culture
- PCR takes a few hours
- detects DNA/RNA therefore not dependent on immune response
PCR vs conventional microbial diagnosis (conventional)
- requires high levels of infecting organisms
- often difficult to distinguish different species
- electron microscopy required to visualise virus
- some organisms cannot be cultured
- culture can take weeks
- hard to be strain specific
- pathogen may not elicit a strong antibody response
how can genotyping the pathogen be used to identify tuberculosis
- conclusive diagnosis depends on detection of M.tuberculosis in sputum
- previously depended on microscopy and culture
- now can achieve same day diagnosis using PCR
phenotyping the disease - what is quantitative PCR
- quantitative PCR measures the abundance of DNA or RNA in a clinical sample for example
- to measure the level of infectious pathogen in a sample
- to measure the level of expression of a gene
how can DNA and cDNA be accurately quantified by real time qPCR
- PCR product is measured as it is produced e.g. by incorporating fluorescent marker into the product
- the cycle number at which the fluorescence reaches a threshold value is measured
- the lower the ct value, the greater the quantity of DNA/cDNA in the starting template
how can qPCR be used in HIV
- measurement of the HIV viral load by quantitative RT-PCR
- useful for monitoring progress of disease and response to drug therapy
why do we isolate DNA
- for genetic manipulations
- for DNA analysis e.g. scientific, medical, forensics, ecological, archeological
what are the steps of DNA isolation
- cell lysis
- DNA purification from the cell extract
- concentrate DNA
- measurement of DNA purity and concentration
what is cell lysis
- release the DNA from the cell by breaking down the cell membrane
biological method of cell lysis
- uses enzymes to disrupt cell membranes. difference enzymes for different cells
- plants - cellulase
- bacteria - lysozyme
- eukaryotic cells - sappanin
physical methods of cell lysis
- osmotic pressure - excess water moves into the cell when cells are placed in hypotonic solution
- freeze-thaw - repeated cycles of freezing and thawing ruptures cell membrane through ice crystal formation
mechanical methods of cell lysis
- grinding e.g. pestel and mortar, bead mill, vortex
what do we not want in our sample of DNA
- protein
- ribosomes
- mtDNA
- lipid
- plasmid
how is DNA purified using phenol chloroform extraction
- lysed cells or tissues are mixed with equal volume of phenol:chloroform mixture
- centrifugation - 2 distinct phases as the phenol:chloroform mixture doesn’t mix with water
- DNA concentration - 0.3M sodium acetate and 2.5 volume ethanol can be used to precipitate DNA from salt and sugar to concentrate it
how can DNA be purified using commercial kits
- column contains a silica membrane that binds DNA in the presence of a high concentration of salt
- impurities such as salts are washed away
- a low salt buffer such as water or 10 mM try-cl
- pH 8.5 is used to release DNA from the membrane and collect out
ads of using commercial kits
- not hazardous
- less time consuming
- results in purer DNA then phenol:chloroform extraction
dis of using commercial kits
- expensive
- small volume
- membrane can only bind a set amount of DNA
steps of silica binding DNA
- lyse cells
- add high salt buffer
- wash with ethanol buffer
- elute with very low salt
how can we measure the quantity and quality of DNA
- UV absorbance
- fluorescence dyes
- agarose gel electrophoresis
- capillary electrophoresis
- diphenylamine method
why is it important to measure the quantity and quality of DNA
- efficient extraction = efficient science
- without a good starting point you will never have good output
- genomic testing would be impossible
-PCR/cloning wouldn’t work - forensic science would be unreliable
what are restriction endonuclease
- enzymes produced by bacteria to protect against viral DNA infection
- restriction enzymes cut the foreign DNA
- restriction - act on specific DNA sequences
- endonuclease - cleave the phosphodiester bond within a polypeptide
why use restriction endonuclease
- to make recombinant DNA molecules
- to cut DNA into defined fragments (DNA fingerprinting and mutation analysis)
how do restriction enzymes cut DNA
- make one cut in each of the sugar phosphate backbones of the double helix at their recognition site in the presence of mg2+
- hydrolyse the phosphate group
- cut ends have a 5’ phosphate
what makes up different types of restriction endonuclease
- cut at specific sequences
- different Res - different cutting sites
- some are blunt ends, some sticky
- recognition sites for restriction enzymes are often palindromic
- 5’ GAATTC 3’
- 3’ CTTAAG 5’
what can restriction endonuclease be used for
- to make recombinant DNA molecules
- to cute DNA into defined fragments
what is star activity
- relaxation or alteration of specificity - chemical/drug intervention
- when reaction conditions differ slightly from the optimum for the enzymes
why might reaction conditions change from the optimum
- low ionic strength
- high pH
- high glycerol concentrations
- presence of mg2+
what are the principles of agarose gel electrophoresis
- polymerised agarose is porous, allowing the movement of DNA
- large towards negative end, travel towards the positive end
- samples enter the gel and migrate according to charge, size and shape
implication of DNA being negatively charged
- migrates to positive electrode
- smaller molecules move more easily through gel than larger molecules
- because of the sugar-phosphate backbone migrates to anode
how can we see the movement of DNA in gel electrophoresis
- visualise with intercalating dyes e.g. Nancy red
how can we determine the size of the DNA fragments
- compare size of product of interest with DNA ladder
- plot a graph log10 and mm band migrated down the gel (use MW ladder to create a standard curve)
- determine size of your products
- graph of log DNA size of the markers by distance migrated creating. straight line
- can then plot the distance the product of interest has travelled on the line and estimate the DNA size
what is genome editing
- is a type of genetic engineering in which DNA is inserted, deleted or replaced in the genome of an organism using nucleases
- enabling specific targeting of sequences within the genome without impacting the rest of the genome sequence
- potential to cure genetic diseases in a patient specific manner
what is CRISPR
- clustered regulatory interspaced short palindromic repeats
-adaptive immune system of prokaryotes - the complex cleaves invading DNA to prevent re-infection by viruses
what are the 3 components systems that make up CRISPR
- cas9 - protein component
- crRNA - RNA component
- tracrRNA - RNA component
how can CRISPR act as an adaptive immune regulators
- against invading DNA/RNA
- invading DNA recognised and cut by cas1-cas2 protein complexes into fragment - protospacer
- protospacers integrated into CRISPR locus located in the bacterial genome
- upon viral reinfection, transcription of the protospacers to RNA is activated which bind to cas9
- cas9/RNA duplex is recruited to complementary sequence on the invading strand of DNA
- cas9 cutes DNA strands creating a double strand break to prevent infection
what is the structure of the CRISPR locus
- transactivating RNA
- operon of cas genes encoding cas protein components
- identical repeat array
- spacer of invading DNA
- the complex formed between the trans activating RNA and the protospacer is the guide RNA which enables selective binding of cas9 to invading DNA sequences
role of the cas operon
- cas operon encodes cas proteins required for DNA cleavage
role of tracrRNA:crRNA form the guide RNA
- the duplex formed between the two RNA species is known as the guide RNA
how do protospacers adjacent motifs enable cas9-mediated DNA cleavage
- 2-8 base pair sequence 3-4 base pairs downstream of the cut site
- cas9 will not cut invading DNA without a PAM site irrespective of cas/gRNA binding
- PAM sequences are not present in the CRISPR locus
- prevents bacterial CRIPSR locus being targeted by cas proteins
what two components make up gRNA in bacteria
- tracrRNA
- crRNA
- linker loop used to link the two
how has CRISPR/cas9 been engineered for biomedical studies
- deposition of the cas9 complex at a desired locus of the genome will enable site specific cleavage through nuclease activity
- the repair of the DNA break by endogenous DNA repair pathways enables specific genomic edits to be introduced
why is correct gRNA design essential for selective CRISPR positioning and DNA cleavage
- the gRNA should contain a photo-spacer sequence upstream of the PAM site
- gRNAs should be selective to a single genome locus to avoid off target effects
what is cellular DNA repair needed for
- the pathways are critical for desired CRISPR-mediated DNA editing
repair mechanisms for double strand breaks
- homology directed repair
- non-homologous end joining
how is genetic drift generated
- when cas9 cleaves DNA, a double strand break is introduced
- homology directed repair or non-homologous end joining function down stream to repair DNA
- this will create the desired genetic drugs
why is DNA repairs by NHEJ error prone
- introduces insertions or deletions into DNA
- impacts gene function
how does HDR enable precise DNA repair
- DNA is precisely repaired using sister chromatid during 5 phase of the cell cycle
how does CRISPR mediate gene knockout via NHEK
- target cas9:gRNA complex to gene of interest
- DSB introduced
- cell repairs the break via NHEJ (error prone)
- indels introduced generating a frameshift (premature stop codons introduced)
- normal gene product not expression
how does CRISPR mediate gene knock in via HDR
- DBS introduced by cas9:gRNA complex
- introduce a template that the cell will use to repair the DSB through HDR
- HDR template requires homology arms on either side of the point of mutation inset
- PAM sites are removed from HR template to prevent re-targeting of region
why is androgen receptor signalling a key driver of prostate cancer
- prostate cancer progression is largely driven by androgen receptor signalling
- current treatment aims to inactivate AR by blocking ligand binding
what are the two CRISPR based studies
- to generate a cas9-expressing prostate cancer cell line to knockout AR
- to create a modified prostate cancer cell line to study function of aberrant forms of the androgen receptor - knock in strategy
how is a cas9 expressing prostate cancer cell line developed
- generate prostate cancer cell line expressing cas9
- to knock out the AR gene
- AR gene locus was targeted by CRSIPR to validate activity of cas9
how are androgen receptor variants studied in prostate cancer
- alternative splicing is principally involved in the generation of AR-VS in response to AR targeting agents, such as enzalutamide
- expression of AR-VS is elevated in advanced disease
how is enzalutamide activated
- loss of the AR LBD creates constitutively active transcription factors that are refractory to enzalutamide
how can using a CRISPR knock in strategy create an AR-V only expressing cell line
- gRNA designed to exon 5 of the AR
- template with point mutation - stop codon
- will block synthesis of full length AR
what should be considered in cell therapy
- efficacy of delivery
- regulatory guidelines
- mosaicism
- gremlin vs somatic
- immunogenicity
- specificity - off target affections
how is CRISPR delivered ex-vivo
- remove cells from the patients/donor
- edit genome
- screen/expand cell popultation
- engraft cells back into patients
how is CRISPR delivered in vivo
- package CRISPR/cas in a delivery vehicle
- deliver to patient
how did successful CRISPR editing of CCR5 in vivo confer HIV-1 resistance
- long term CCR5 disruption was observed
- CCR5 disrupted HSCs were able to reconstitute a functional immune system
- viral titre reduction and increase in CD4+ T cells demonstrated HIV resistance
why sequence genomes
- blue print to life e.g. all genes, regulatory sequences, higher order structure, chromosome maintenance, comparative searches
what are the issues with sequence genomes
- cost and scale
- technology
how is an organisms genomic sequence obtained
- obtain the organisms genomic DNA
- break the DNA into small fragments
- obtain the DNA sequence from all the fragments
- search for overlaps to identify between the DNA sequences of the different fragments to reconstruct the genome sequence
- fill in any missing gaps in the genome sequence
what is the importance of model organisms
- small genome - value for money
- easy organisms to manipulate
- provide information on fundamental biological processes
- technology development
what are the major issues identifying genes with genomes
- how big is a valid open reading frame
- identification of RNA splice sites
- RNA analyses can help but depends on the expression of the gene
why problems in gene identification emphasised by genome analysis in s.cerevisae
- it is smaller than the human genome
- genes are tightly packed with very little repetitive DNA
- complete absence of RNA splicing to complicate gene identification
- simple genetics can be used to analyse potential gene function
what can be predicted from compute analyses of protein sequence
- prediction of function - roles for model organisms
- prediction of protein localisation
- prediction of protein domains/modification
what are the benefits of studies in model organism
- functional characterisation of mutant proteins
- understanding human genome variation
what is an example of identification of functional characterisation of mutant proteins
- analysis of predicted catalytic mutant Msh2 proteins from human colon cancer was confirmed by expression the proteins in yeast
what did analyses of other mutant Msh2 proteins reveal
- defect in critical protein-protein interaction
- reduced steady state levels of Msh2
- mutations affected the activity of the mismatch repair complex
example of the importance of cellular localisation
- cellular localisation of the schizosaccharomyces pome cell cycle regulatory Yox1
- analyse the protein using the PSORTII programmed
- yox1 localises to the nucleus in all stages of the cell cycle in schizosaccharomyces bombed
example of the importance of prediction of protein domains/modifications
- regulation of the schizosaccharomyces pome cell cycle regulator yox1
- to understand function/regulator of the protein use a range of programmes to predict potential functional domains and protein modification sites
how can yox1 be analysed using domains
- use various programmes including BLAST to identify conserved domains
what was discovered in the domain analysis of yox1
homeodomain - DNA binding domains involved in the transcriptional regulatory of key eukaryotic developmental processes; may bind to DNA as monomers or as homo and/or heterodimers, in a sequence-specific manner
what is phosphorylation site analyses of yox1
- search for potential serine/threonine/tyrosine phosphorylation sites
- for example using NetPhos programmed
what approaches can be taken to undergo phosphorylation sites analyses of yox1
- investigate protein phosphorylation in vivo and if so whether the identified threonine residue is important
- test genetically and biochemically the potential role of the programme predicted kinase
- mutate the threonine residue to a glutamic acid, an aspartic acid or an alanine residue to investigate role of phosphorylation
what are the uses of genome sequence within an organism
- identification of regulatory sequences
- characterisation of protein families
what are regulatory sequences
- identify all promoters containing a transcription factor binding site
how are protein families characterised
- kinases have well characterised homology within catalytic domains
- genome analysis allows inference of the function of uncharacterised kinases by family studies
- genome analysis allows identification of conserved and organism-specific families of protein kinases
what are the different types of study for genomic experiments
- protein/DNA interactions
- DNA methylation
- gene expression
- protein-protein interaction
- loss of function
what are microarrays
- many functional genomics experiments depended on microarrays
- measurement of hybridisation
- sample to probes on array
- range of samples and probes for different experiment types
steps of microarray
- extract RNA
- reverse transcription
- cDNA
- in vitro transcription
- biotin labelled cRNA
- random fragmentation
- fragmented biotin labelled cRNA
- cRNA hybridisation to gene chip
- wash away non-specific binders and stain with streptavidin-phycoerythrin
- scan array with laser, detect fluorescence with CCD, read image into computer
how did we go from microarrays to sequencing
- direct sequencing can substitute for hybridisation
- give greater resolution and accuracy
- issues were cost and throughput
- development in sequencing have addressed both
what is high-throughput sequencing
- refers to range of technologies
- competition - driven down cost and increased throughput
- illumina sequencing dominates
what is illumina sequencing
- fragments of DNA bound to solid surface
- binding enables by special sequences ligated to fragments
- solid-phase bridge PCR forms clonal clusters
- sequencing proceeds in cycles
- modified nucleotides with fluorescent group which blocks extension
- reversible termination allows sequencing to proceed to next cycle
what is RNA sequencing
- use of high-throughput sequencing technologies to get information about a samples RNA content
- mRNA are converted to cDNA
- cDNA used for sequencing library generation
- allows quantification, profiling and discovery of RNA
how does RNA sequencing work
- polyA selection
- fragmentation
- random printing
- first and second strand cDNA synthesis
- end repair, phosphorylation and A tailing
- adapter ligation, PCR amplification and sequencing
how does amount of RNA effect RNA sequencing
- sequences in final library are derived from RNA population in sample
- presence is proportional to original sample - more abundant RNA species will be present more frequently in library
- random priming is attempt to remove bias
- actual randomness debatable
what are the consideration for RNA sequencing
- big data sets require expert processing
- expression data can be noisy
- easy for confounding factors to dominate
- good practise same as for any statistical approach
what are the other functional genomics approaches
- many assays of aspects of nucleic acid biochemistry based on sequencing
- general idea is to enrich specific molecules or regions of molecules and sequence
- then analyse to show what you’ve enriched
what is ChIP-sequencing
- cross link proteins to DNA
- isolate DNA and shear
- immunoprecipitate protein of interest
- reverse cross-linking
- purify DNA
- sequence
what is ATAC-sequencing
- assay for transposes accessible chromatin
- similar to older DNAse-sequences
- relies on transposase Tn5
- adapter ligated fragments isolated, amplified and sequenced
what are the feature of transposase Tn5
- high activity transposase
- highly efficient cutting of exposed DNA
- ligation of adapters to ends
what is bisulphite sequencing
- bisulphite treatment is used to determine the methylation state of DNA
- methylated cytosine protected from deamination
- unmethylated cytosine converted to thymine
what is the importance of reduced representation BS-seq
- RRBS utilises Mspl restriction enzyme to enrich for CpGs
- results in fragments which begin/end with CpG
what are the key point of functional genomics and bioinformatics
- high through-put sequencing produces large amount to data
- data can be noisy and complex
- computational approaches needed to make most of data
what are the main examples of model organisms
- mouse - mus musculus
- clawed frog - xenopus sp.
- zebrafish - danio rerio
- fruit fly - drosophila melanogaster
- nematode worm - caenorhabditis elegans
what are the key point of mice for model organisms
- mice are an extremely well characterised model organism
- we share most of our genes with mice
- mice have a short lifecycle - useful for rapid breeding of new stains
- however, they are relatively expensive to keep
what are the 3 licenses required for animal experiments
- personal license for the researcher
- project license for the study
- establishment licence for the place where the study is carried out
why aren’t in vitro models commonly used
- not representative of a proteins role in a biological system like a body
- different cell types interact with each other constantly within living tissues
- these interactions are impossible to model outside of animals
what are the different mouse models used to study human development and disease
- transgenic mice
- knock-out mice
- knock-in mice
what are the step in creating a standard transgenic mouse
- gene is microinjected into the pro-nucleus of a fertilised mouse oocyte
- injected oocytes are transferred to a pseudo-pregnant recipient mouse
- all offspring are screened for expression of the trans gene by DNA analysis
what are the stages in creating gene-target transgenic mice
- an isogenic tranigen with a drug selection gene is introduced into embryonic stem cells
- drug selection is used and surviving cells are screened for the correct integration of transgene
- correctly targeted cells are microinjected in mouse blastocysts
- blastocysts are transferred to pseudo-pregnant recipient mouse
- chimeric offspring are identified and mated to test for gremlin transmission of trans gene
what is required for simple vector construct (transgenic approach)
- gene of interest
- relevant promoter
- 3’ protein tag for detection
- poly A tail
what are the benefits of transgenic mouse models
cheap
easy to make
wild type gene prod is still present - can express human genes in mice
what are the drawbacks of transgenic mouse models
- multiple founders are generated - need to characterise numerous mouse lines
- can not control site of integration into genome
- wild type gene product is still present
drawbacks and benefits of gene targeting approach for knock out mice
- precise
- requires a complex vector and relies on homologous recombination between vector and host genome
what are the stages in gene targeting screening
- electroporation of targeting vector into ES cells
- establish homologous recombinant ES cell closed by 1st screening, PCR analysis, southern blot analysis
- production of chimeric embryos by aggregation method
- transfer to recipient mice
- obtaining chimeric mice. contribution of ES cells is estimated by coat colour
- crossing chimeric mice with wild-type mice to obtain F1 heterozygous mice
what is gene trapping
- a high-throughput approach that is used to introduce insertion mutations across the genome in mouse embryonic stem cells
what are the consequences of the insertion of a gene trap vector
- disrupts gene function
- reports gene expression
- provides a convenient tag for the identification of the insertion site
what is the international gene trap consortium
- administers all publicly available gene trap cell lines
- researchers can search and browse the IGTC database for cell lines of interest
what is the relevance of genetrap for mouse phenotyping consortium
- to provide targeted inactivation through recombination using FRT and loxP
what certain genes inactivate gene expression
- lacz - to localise gene expression
- neo - for antibiotic selection
what is the importance of FRT and loxP
- FRT and loxP sites mediate site specific recombination through DNA recognition sites
what is cre recombinase
- is a tyrosine recombinase enzyme derived from p1 bacteriophage and catalyses site-specific recombination between two loxP sites
what is flippase
- is a tyrosine recombinase enzymes derived from the bakers yeast - saccharomyces cerevisae - and catalyses site specific recombination between two FRT sites
what is the importance of loxP
- loxP sites allow straight forward knock-outs to be generated
what is the cre-lox system for conditional alleles
- following manipulation the mouse has a targeted but flowed allele
- a second mouse is transgenic for core recombinase expressed under the control of a tissue specific promoter
- the two mice are crossed together to generate a mouse line that carries both alleles: floxed and cre
what is the result of the crd-lox system for conditional alleles
- tissue specific deletion of the floxed allele only in tissues where cre recombinase is expressed
what are the benefits of knock out mouse models
- gene traps available for virtually all genes from international mouse phenotyping consortium
- can make conditional KO using cre recombinase
what are the drawbacks of knock out mouse models
- can stress or cross onto a different background
- may not accurately model a known human disease –> loss of function vs dominant negative
when is knock in technology commonly used
- used to introduce a human mutation into the mouse genome
what are the benefits of knock in mouse models
- genetically relevant mouse model of a human disease
- crisp/cas 9 has lowered cost and time
what are the drawbacks of knock-in mouse models
- traditionally time consuming and expensive
- remaining loxP site may be a problem - might interfere with mRNA stability and/or expression
what is CRISPR-cas9
- CRISPR is a form bacterial adaptive immunity
- it utilises short RNA molecules in concert with a DNA cutting enzyme to protect against viral infection
- it has recently been repurposed by researched to edit DNA in vitro
what are the examples of disease modelling using a variety of mouse transgenics
- the genetic skeletal diseases
what did the mouse model of a genetic skeletal disease
- short limb dwarfism phenotype
- mutant protein retained in ER of chromosomes
- reduced cell proliferation
- increased spatially dysregulated apoptosis
