Animal Models Flashcards

Question

Describe CRISPR

Answer 1

CRISPR - origins in bacterial adaptive immune system Associated with Cas9, an endonuclease that causes ds-breaks, which binds an RNA enabling specificity, targeting sequences ending in NGG (PAM sequence) Cas9 causes ds-breaks 3 bases upstream of the PAM sequence There is 2 ways of repairing breaks = non-homological end joining, or homology directed repair NHEJ = error prone = purposefully creating mutations = KO HDR = used to add in DNA sequences from donor DNA but less efficient = knock-in

Answer 2

Ease and low cost at raising large numbers Highly conserved genetic and biochemical pathways between ZF and mammals Wildtype/transgenic screens - target organs/systems Morpholino or mutant rescue screens Drug reprofiling and FDA approved libraries Automated screening is now available

Answer 3

RAS encodes GTPases most commonly activated in cancer Injection of human RAS causes early lethality HRAS was put inserted into ZF under a heat shock promoter Heat shock leads to more HRAS which showed via microarray that it caused upregulation of dusp6 This upregulated version was used to screen for drugs that can inhibit the upregulation

Answer 4

Screened 5648 compounds for changes in multiple parameters: rest bouts, rest latency, waking activity 547 drugs significantly affected behaviour Assigned behaviour fingerprints to each compound (reveals relationship between drugs and their targets

Answer 5

Prior to public availability it was originally a sedative used for many symptoms including nausea Research found It was almost impossible to give a lethal dose Testing: Thalidomide bound to ferrite beads, incubated with HeLa cell lysate Beads with bound protein washed, elution of bound proteins with free thalidomide SDS gel electrophoresis and silver staining, mass spectroscopy to identify proteins that bind thalidomide interacting proteins identified - cereblon and damaged DNA binding protein 1 (DDB1) Cereblon KD causes thalidomide-like defects on ZF Injection of cereblon that doesn't bind thalidomide rescues effects of thalidomide

Answer 6

Many genetic models are also good disease models, but: Replicating a human genetic defect doesn't necessarily create a disease model Generating a functional defect does not necessarily replicate the human genetic disease Creating a disease model can require more than simple genetic modification – e.g. “humanization

Answer 7

Although many genes have highly conserved sequence and function, there are differences Genetic background makes a difference Gene dosage can have different effects between human and mouse Complex phenotypes are difficult to replicate e.g. neurobehavioural defects

Answer 8

Transgenesis - stable insertion of exogenous DNA into host cell’s chromosomal DNA - Doesn't include transient transfection Exogenous DNA with the same sequence as endogenous DNA can lead to exchange: known as homologous recombination Genome engineering techniques use modified nucleases to cut, or nick genomic DNA Endogenous DNA repair enzymes cause mutations or can be directed to insert novel DNA sequence

Answer 9

Embryo manipulation is important for transgenesis to generate a germ-line genetic modification so mutation can be passed on

Answer 10

DNA microinjection into zygotic pronucleus Relatively quick & easy Transient assays possible (we can look at embryos, for example)

Answer 11

Targeted - Homologous recombination in embryonic stem (ES) cells Relatively slow and difficult Very powerful and flexible Targeted - Genome engineering techniques in ES cells or embryos Ease and speed depend on the technique used Possibility of off-target effects

Answer 12

Fertilisation - large male pronucleus injected into egg, but doesn't fuse with the female immediately Zona pellucida constrains the growth of the early embryo After 32 cells, there starts to be a distinction between two cells - trophectoderm , and the blastocyst containing the inner cell mass Trophectoderm mainly create external embryonic tissue

Answer 13

4 week old females are superovulated and mated → fertilised eggs are collected and allowed to mature until the male pronuclei starts to move to the centre of the zygote DNA is microinjected into the male pronucleus (200-400 eggs/day, 75% survival) → embryos are transferred into the oviduct of pseudo pregnant females (25-35 embryos per mouse) 18 days later pups are born (25% success rate) → screen DNA to identify transgene incorporation (10% success)

Answer 14

For disease models, most commonly trans-speciation e.g. human gene in mouse Repression of a gene - shRNAi (or morpholino antisense) inhibition of expression (Over)-expression of a gene “Normally” – its own promoter “Ectopically” – another promoter Express in different cell types, different times, and Cre Reporter of gene expression - attach easily detectable protein/enzyme Promoter or non-coding region analysis Pronuclear injection is also used in genome editing

Answer 15

The transgene contains promoter, enhancer, cDNA, some 5' and 3' UTR, 3' intron and polyA signal Important to distinguish the transgene from the endogenous gene You can find the expression by Promoter can be changed - ubiquitous/tissue specific, inducible and vary length of promoter/enhancer Gene - what are its properties, gene from which species, attach tags (epitope tags or reporter)

Answer 16

Promoter traps - constructs containing a reporter gene and selectable marker but no promoter Splice acceptor at 5' end of gene biases for integration into 'active' regions of the genome E.g. Gene = beta-galactosidase (lacz) and neomycin resistance Thus the protein is only expressed when it is successfully integrated into DNA under a promoter, thus the protein expression pattern can be observed to learn about it

Answer 17

Similar to promoter traps but instead you're focusing on the gene - constructs containing a reporter gene and selectable marker but no promoter Splice acceptor at 5' end of gene biases for integration into 'active' regions of the genome This is transfected into embryonic stem cells growing in culture Reporter gene has a selectable marker so only transgenic cells are left These cells are then implanted into a blastocyst which is implanted into a female mouse Create chimeric mice as the edited stem cells will proliferate at different levels compared to others Germline-transmission is then checked from its offspring

Answer 18

Pluripotent cells isolated from the inner cell mass cells of a blastocyst They proliferate indefinitely in culture (and remain pluripotent, under the right conditions) Can be genetically manipulated in culture Form the basis for the vast majority of targeted transgenics

Answer 19

Homologous chromosomes pair up, create 2 ds-breaks forming a Holliday junction Reminder - this can create CNV's (non-allelic homologous recombination) This processes causes swapping of a group of alleles However, the repair enzymes only recognise the end regions Thus the middle segments can be altered DNA can be introduced between regions identical to host DNA However, homologous recombination occurs at low frequency Positive drug selection is needed to identify these rare events DNA integration can also occur at random locations These can also confer drug resistance/sensitivity, allowing negative drug selection To select for this the gene would be at the END of the recognised sequence, so it would not undergo homologous recombination and only be integrated through this mechanism You want to prevent this so you kill these cells of using a drug this gene causes sensitivity to

Answer 20

As homologous recombination is rare, we need to be able to select the ES cells in which it has taken place Positive selection - include gene in the recombination region, conferring resistance to a toxic drug Negative selection: include gene in the construct that confers sensitivity to a toxic drug Outside of recombination region, to kill cells in which random integration has occurred (gene at end) Positive selection: (PGKneo bpA, neo) - neomycin phosphotransferase, makes cells resistant to G418 Use phosphoglycerate kinase promoter and bovine growth hormone poly A tail Can also use hprt and HAT medium Negative selection: (MC1TKpA, tk) - Herpes simplex virus thymidine kinase, makes cells sensitive to ganciclovir or FIAU

Answer 21

2 transgenes, one making a drug responsive repressor or activator protein The other has a response element with the gene of interest

Answer 22

Four amino acid changes to TetR (repressor) alter its binding characteristics and create reverse TetR (rtTA) - thus becoming an activator This binds the TRE (tet response element) in the presence of doxycycline and activates transcription

Answer 23

The tet-controlled transcriptional activator (tTA) is a fusion of the wild-type Tet repressor (TetR) to the VP16 activation domain (AD) of herpes simplex virus tTA binds the Tet-responsive element (TRE) and prevents transcription in presence of tetracycline or doxycycline

Answer 24

This is a starting point for inducible gene expression Wild-type TetR is a repressor protein from bacteria - a basis for antibiotic response in bacteria When present in the same cell TetR will bind the TRE and turn OFF gene expression TET/DOX binds TetR, it can’t bind to the TRE = turns ON gene expression Thus by default (no TET) gene expression is off Binds TRE and turns off expression in absence of Tet/dox Cannot bind TRE and activates transcription in presence of Tet/Dox

Answer 25

TetR is modified to add the Trans-Activation domain from VP16 (Herpes Simplex Virus) = tTA This tTA protein binds to the TRE and turns ON gene transcription in the absence of Tet TET/DOX binds tTA, it can't binding to the TRE = turns OFF gene expression Thus by default (no TET) gene expression is on Binds TRE and activates transcription in presence of Tet/Dox

Answer 26

TetR is modified to add the Trans-Activation domain from VP16 Thus by default, without binding it now deactivates promoter AND 4 amino acids are altered to generate rtTA (reverse tTA) Prevents TRE binding in the absence of antibiotic (Tet) keeping gene expression turned OFF Tet permits rtTA binding to the TRE = turns ON gene expression Thus by default (no TET) gene expression is off Binds TRE and activates transcription in absence of Tet/Dox

Answer 27

Normally the estrogen receptor (ER) is in the cytoplasm, complexing with HSP90 (heat shock protein) When oestrogen diffuses into the cytoplasm oestrogen binds the ER It then dissociates from HSP90 ER then travels into the nucleus to dimerise with other ER and drive transcription of downstream target genes This system can be adopted by taking the ER ligand binding domain, fusing it to another protein (TF) so that the protein/TF will only turn on its target in presence of oestrogen A reporter gene can be placed downstream of the ER-responsive element

Answer 28

The creation of a null (completely non-functioning) mutation for a specific gene Sometimes used to model a human loss of function mutation/disease Usually done by removing all coding exons, or essential exons near the 5’ end of the gene The gene is removed in all cells of an embryo, from the beginning of development Many null mutants are lethal Some null mutants have no effect (redundancy)

Answer 29

Inactivating mutations of the CFTR introduced by insertion of exogenous gene sequences CFTR DF508 mutations introduced by homologous recombination CFTR null mice have a more severe phenotype than DF508 mutations Both mutants die shortly after birth BUT from intestinal obstruction, not lung problems! Specific strains of Tg mice show some lung pathology under specific conditions

Answer 30

Many gene knockouts are (prenatal) lethal, so there is the need to control gene deletion, using recombinases to create conditional knockouts ``` Cre is a site-specific recombinase with a 34bp recognition site (loxP) 2 inverted (palindromic) repeats and core GCATACAT ``` Cre recombinase induces recombination between two loxP sites, in cis (same Chr), or in trans (diff Chr) Effect depends on the orientation of the loxP sites Usually used to remove intervening sequence between the loxP sites Image in notes shows 3 possible loxP deletion patterns ``` A = cis/homologous Chr, same strand - cut out, forms circle which is degraded (colour image) B = cis/homologous Chr, different strand - translocation C = trans/non-homologous Chr - translocation ```

Answer 31

Cre is not the only recombinase Cre and Flp are the most widely used recombinases and others (eg Dre, PhiC31) Uses frt sites instead of loxP for Flp The principle is the same - a short recognition sequence, a separate, site-specific recombinase

Answer 32

The region of genomic DNA is cloned into bacteria and modified then used to generate targeted ES cells by homologous recombination to generate the loxP site Insert loxP sites into the introns, as far away from exon-intron boundaries, to flank the exon to be deleted This is to avoid splice site interference Targeted ES cells are used to generate a mouse line with a “floxed” (flanking loxP) allele These mice are normal Cross the floxed mouse with a Cre mouse (this Cre mouse is a separate transgenic animal) Offspring thus has that cre that removes the flanked allele = target exon deleted Expression of Cre in the cre mouse can be driven by different methods Knocked in to an existing gene, so controlled by that gene’s promoter Driven by a promoter of its own – this could be expressed in all tissues, or some and expressed all the time, or at specific times e.g. combined with Tet Can be turned on/off by specific drugs that are given to the mouse e.g. tamoxifen Wherever Cre is expressed, it recognises the loxP sites and recombination removes the floxed exon Once recombination has occurred, that DNA is totally deleted

Answer 33

Notes = Images Insert flanking loxP Insertion of neomycin (antibiotic resistance) flanked by frt Use neomycin to select cells that underwent homologous recombination You can then remove it with Flp the frt sites by crossing with Flp mouse Mouse > add sequence that flanks the exon target and insert frt flanked neo > select successful via neomycin > cross with Flp mouse to remove neomycin gene > cross with Cre mouse to remove exon

Answer 34

A fusion protein of Cre-ERT is used (expression driven in all tissues or in specific cell types) ERT is a modified version of the ligand binding domain of the oestrogen receptor with high affinity for Tamoxifen (= lower affinity for endogenous oestrogen) ER is normally held in the cytoplasm by Hsp90 Addition of Tamoxifen causes Hsp90 dissociation and the Cre-ERT protein translocate to the nucleus where Cre mediates loxP recombination

Answer 35

Notes = image ``` Gene: Exon 1 Selection marker - neomycin resistance gene flanked by frt & loxP1/2 Reporter gene - lacz is flanked by frt Exon 2 - flanked by loxP 2/3 ``` Due to the large amount of exogenous DNA, the gene is inactivated = KO FIRST Route 1: Select for recombination FLP = removes selection marker + reporter gene Cre = removes exon 2 Route 2: Select for recombination Cre = removes resistance gene nd exon 2 Only reporter gene remains

Answer 36

The vast majority of models are mouse models, and until recently, all made by transgenesis – that is the introduction into the mouse genome of “unnatural” DNA, artificial or from another species Recently, genome editing technology has allowed changes to be made directly to genomic DNA sequences

Answer 37

Zinc finger TALENs CRISPR-Cas9

Answer 38

Protein with a DNA cutting enzyme and a DNA-grabbing region Can be programmed to recognise different genes Off target cuts are a problem - not very precise

Answer 39

Protein with a DNA cutting enzyme and a DNA-grabbing region Can be programmed to recognise different genes - easier to design than zinc finger Off target cuts are a problem - not very precise

Answer 40

DNA-cutting protein (cas9) guided by an RNA molecule Cas9 works on any sequence of guide RNA as long as it has a tracer RNA Cas9 recognises the trcrRNA and binds RNA and DNA nduces ds-DNA breaks at a location Cellular machinery tries to repair the clean ds-break by NHEJ Guide RNA can add a homologus DNA sequence as a template to repair the break, thus inserting the donor template (HDR) More precise, but less efficient Affordable, easy to use and useful in high-throughput multi-gene experiments Can make off-target cuts - but more specific due to guide RNA

Answer 41

Cas9 has off-target effects Random nuclease activity can occur throughout the genome and generate non-specific effects/mutations Modified versions of Cas9 have fewer off-target effects Nuclease can be modified to have a nickase activity - so it cleaves only one strand at a time By targeting the two separate strands, you can minimise the off-target effects as you need a ds-break Cas9 can be modified into an activator, repressor or reporter Also modifications with altered PAM specificity allow for more precise targeting

Answer 42

Affects face, heart, neural tube, thymus and parathyroids Two common breakpoints - typically deleted regions and DiGeorge critical region Involves intra-chromosomal recombination at low copy repeats - mediated by homologous regions The Df1 Mouse Mutant A multigene deletion of a homologous, syntenic region of 22q11 Df1/+ mice have 22q11DS-like aortic arch defects 22 genes are deleted including Tbx1 Complementation (rescue) and single gene KOs identify Tbx1 as key gene for pharyngeal phenotypes

Answer 43

TBX1in 22q11 Deletion Syndrome Tbx1 is expressed in the pharyngeal region Tbx1 mutations in animal models phenocopy many physical defects of DGS BUT, heterozygous Tbx1+/- mice have a transient, embryonic phenotype Both copies of Tbx1 have to be inactivated to produce the full spectrum of human phenotypes