Module 08- System models PDF

HSS 3109 - Research Approaches in Health Biosciences Yan Burelle, Ph.D. Professor Interdisciplinary School of Health Sciences, Faculty of Health Sciences & Department of Cellular and Molecular Medicine, Faculty of Medicine University Research Chair in Integrative Mitochondrial Biology University of...

HSS 3109 - Research Approaches in Health Biosciences Yan Burelle, Ph.D. Professor Interdisciplinary School of Health Sciences, Faculty of Health Sciences & Department of Cellular and Molecular Medicine, Faculty of Medicine University Research Chair in Integrative Mitochondrial Biology University of Ottawa Pavillon Roger Guindon Room 2117 451 Smyth Road, Ottawa, Ontario K1N 8M5 Lab website : www.burellelab.com Phone (office) : 613-562-5800 ext 8130 System models: focus on laboratory mice • Understand what are Transgenic mice and how they are generated • Understand what are Knockout mice, how they are generated and their interest • Understand what are Knock-in mice, how they are generated, and their interest • Understand what are inducible and tissue-specific models and what are their interest System models: focus on laboratory mice Mouse is the best preclinical model due to these advantageous features Animal facility - each rack has 80 cages and in each cage, you can put 6 mice Easy to modify the genome of their embryonic stem cells Small Can house a lot of animals in a restricted place However, in closely related species like rats, hamsters, etc. it is not as easy Short gestation time Pups every 21 days Produce large litters Up to 6-8 pups per litter, sometimes up to 11-12 Homology with human genes* (88% homology of coding DNA) *Non-coding DNA differs substantially Trangenic mice C57BL/6-Tg(UBC-GFP)30Scha/J • Transgenesis is the addition of foreign genetic material in the genome of an organism. Inserting gene that does not exist in organism • Transgenesis in mice was successfully performed in the early 1980s One of the first transgenders produced: Green fluorescent protein GFP GFP gene introduced in pronucleus of fertilized oocyte ; fluroscees green form the get go • It has now become a major research tool in health biosciences: • Label proteins & cellular structures (ex: GFPtransgenic mouse) • Study of a protein its fate or biological function • • ex. Can overexposes a protein present in body to see impact of having more of this protein introduce mutated protein to see impact Mice are bright fluorescent green; can visualize their structure in quite deep detail w/ ex. Fluroescence microscopy Knockout mice • Trangenesis à introduction of a new function performed by a foreign gene • Knockout à disruption of an existing function by blocking a specific gene Inactivating a gene that is expressed and functional in the mouse • Knockout = Loss of function Can study impact of loss of function on phenotype • Transgenesis = Gain of function • Genetic knockout is important for understanding the physiological role of a specific gene in an organism Hormone: regulates appetite in the brain • knockout: remove “brake” on appetite and mice start eating like crazy and become super fat Leptin-knockout mouse vs wild-type Methods to introduce a transgene or create a knockout in mice Three approaches can be used: Most commonly used approach Retroviral approach DNA injection Plasmid w/ gene of interest is microinjected Microinject This step is earlier than 8 cell stage; it is just after fertilization transgene Produce pups that have incorporated the trans gene at various levels Embryonic stem cell approach Not all of them, but some of them Workflow to generate transgenic mice with the ES cell method Step 1: Introduce transgene in embryonic stem cells Step 2: Generate chimeric embryos Cross another set of parents together Cross male w/ female Generate ESC w/ genes that reduce a black coat • have genes for white coat Take out fertilized embryo In trophoblast Some ESC will incorporate cloned gene and start to overexposes it ESC are there. Trophoblast has formed. Extract ESC and put in culture • Electroporate vector in ES cells • Establish homologous recombinant ES cells by 1st screening, PCR analysis and these to select cells that have Southern blotting Use incorporated the plasmid at proper • Production of chimeric embryos by aggregation of ES cells place • has DNA from donor parents, and some coming from the ESC donor A genetic chimerism or chimera also chimaera is a single organism composed of cells with distinct genotypes. Workflow to generate transgenic mice with the ES cell method Step 3: Generation of chimeric mice by microinjection of chimeric embryos Laborious process, typically takes 2 years to generate the mouse line Treated w/ hormones to make uterus receptive for better implantation of embryo • Implant chimeric embryo into the oviduct of a pseudo-pregnant shows highest percentage of recipient female transgenic cells • Obtain chimeric mice (contribution of TG ES cells is estimated by coat color) 19-21 days later first litter of pups F1 generation Only 1 allele (donated by the chimera) contains the transgene F2 generation Litter mate controls • genome is identical except for genetic modification of the specific gene Last two mice have Progeny more black than white: proportion of (chimeric mice)modified gene is ES cell donor (C57Bl6 strain) ¼ of the progeny will have 2 transgenic alleles After second generation of breeding, you end up having homozygous genetically modified mice, and can get wild-type controls form the same litter Host parent (CD1 strain) greater in these pups compared to the rest • best ones to create your n ew mouse line Strategies to create a Knockout mouse model • Several methods exist to disrupt the expression of a gene • The classic approach is the one that uses homologous recombination • • Homologous recombination Insert neo-cassette in critical part of gene (exon 2 in this case) Inserted into plasmid normal process that occurs in our genome exploit process to insert construct that will interrupt the expression of particular gene Sequence of gene you want to knock-out • For this, a Neo cassette (Neor) is introduced in the gene of interest at any important coding region that is essential for a gene’s function. l Critical region • Improper placement of the Neo cassette can however result in an incomplete knockout of the gene of interest When you copy gene, it will stop at neocassette and generate the truncated mRNA and no protein, resulting in knocked-out expression • Alternative or cryptic promoters that are not disrupted in the targeted allele • Differential splicing or Read-through transcription of the drug resistance gene can generate RNA species that has some coding sequence from the targeted allele Strategies to create a Knockout mouse model • The first step is to culture embryonic stem cells (ES cells) derived from mouse trophoblast • The second step is to introduce a replacement vector in these ES cells, and select those in which homologous recombination has occured Target vector w/ neocassette Strategies to create a Knockout mouse model i.e. exon flanking the insertion • This is done in a two step selection procedure using: • The antibiotic Neomycin (requires the Neor cassette) • The DNA synthesis inhibitor Gancioclovir (requires the HSV-tk gene) The Neomycin-resistance cassette is introduced between 2 exons of the targeted gene, while the thymidine kinase gene from HSV virus in added beyond the sequence of the target gene. Tymidine kinase gene driven by a strong promoter from Herpes Simplex Virus (HSV) Strategies to create a Knockout mouse model • Two step selection: • Positive selection: selection with the neomycin antibiotics will kill any non-recombinant ES because they lack the Neomycin resistance cassette (NeoR) • Negative selection: Selection with ganciclovir will kill the non-homologous recombinants carrying the TKHSV gene which confers sensitivity to ganciclovir, a cytotoxic nucleotide analogue When treated w/ ganciclovir, TKHSV gene produces nucleotide that is toxic for cells; prevents DNA synthesis and nucleotide is not properly incorporated in DNA Homologous regions will recognize exons on endogenous gene and insert neocassette in proper place will generate knockout • HSVTK gene eliminated Regions of homology: blue - have neocassette in btwn them Pink: HSVTK gene • transfect ESC w/ this vector Not at proper spot • no knockout of gene Knockout mice • In this example a targeted germ-line deletion of exons 4–7 of the PINK1 gene is shown Knockout strategy for the PINK1 gene ECOR1 restriction site • Deletion of exons 4–7: 1) removes the majority of the kinase domain and 2) creates a nonsense mutation (i.e. introduction of a stop codon) at the beginning of exon 8 because of a shift of the reading pol will detach and transcription will stop there frame. DNA • if exon 8 is there, wild-type; if not, it is a knock-out Exon 4-7 replaced by neocassette Exon 3 and 8 are the homologous regions selected no protein is synthesized Forward and reverse II Check if neocassette is inserted in the right place Primers P3 and P4 are used to Note: in this model HSV-tk is replaced by PGKconfirm the correct recombination DT (Diphteria-Toxin gene driven by the in the 5ʹ homologous region of ES PhosphoGlycerate Kinase promoter) for cells. Those are the cells that will negative selection of random insertion (see be used to generate the chimeric previous slide) Only generate fragment if is properly mouse neocassette placed btwn exon 3 and 8 Extra test on top of antibiotic selection steps Same as TK gene PGK gene eliminated Has exon 1-3 but lacks everything downstream • The truncated mRNA from the targeted allele is likely to be degraded through nonsense-mediated decay. I III used to screen ES cells to identify those that have incorporated the targeting vector prior to injecting them into embryos Primer PI and P2 are used to amplify the 3ʹ homologous region that contains exon 8. This is used to confirm knockout in mice Knockout mice Knockout strategy for the PINK1 gene Southern blot analysis of ES cells DNA prior to injection in embyos Detected using the probe specific for the 3′ probe which amplifies a fragement flanked by the EcoRV site (E on the lef map) and the 3I One allele. Normal and probe) other is truncated Wild type allele = 67.1 kDa is much Targeted allele = 6.9 kDa Fragment shorter as its lacking Two alleles have no insertions and only wild type is amplified We never get -/- in ES cells exon 4-7 Northern blot analysis of mRNA in F2 mice Amplified under 2.5 kb Using primers (P1 and P2) detecting exon 8 . This exon is degraded in the knockout mouse because it is tuncated and unstable Knock-out: both alleles are messed up Middle: optical density of band is diminished vs. Wild type Knockout mice • In addition to homologous recombination several other methods now exist to disrupt the expression of a gene: • Site specific nucleases have been recently developed: • Zinc Finger Nucleases • Transcription activator-like effector nucleases (TALENs) • Clustered regularly interspaced short palindromic repeats (CRISPR) and Cas9 protein CRISP-Cas9 • The CRISPR (Clustered Regularly Interspaced Palindromic Repeats) system is the equivalent of our adaptive immune response for bacteria: • It allows bacteria to recognize a virus that has previously infected the and • Specifically target and destroy regions withing the viral DNA making it inneffective Acquisition: Viral DNA sequences adjacent to recognition motifs are inserted in the bacterial genome and forms a CRISPR region Tracr RNA shRNA Infection: Viral DNA goes in bacteria Destruction: Viral DNA is destroyed, preventing viral replication crRNA to make sure it does not incorporate in bacterial genome and mess it up • The system makes use of: • Cas9 (a double strand cutting endonuclease) • Single stranded guide RNA (Tracr+crRNA) Cas9 maturation: In case of a new attack, Cas9, transactivating RNA and the specific CRISPR guide are transcribed and form a complex Recognition: Cas9 recognize PAM sequence located nearby the viral DNA region complementary to CRISPR New infection from same virus previously encountered CRISPR-Cas9 For genome editing Single guide RNA • A single guide RNA is designed to recognize a specific region of the genome (usually 18-20 nucleotides long) • This sequences needs to be close (10Short motif that CAS9 recognizes 15 bp) to a PAM region for Cas9 to work (but there is one PAM every 42 base pairs…so its not a problem!) so CAS9 can pretty much bind everywhere on the genome • The Recognition (REC)Cutsand Nuclease lobes (NUC) of Cas9 then nick the two strands of DNA Allow to attract Cas9 to the site RNA sequence that you can design to be complementary to a region in genome you want to edit to direct CRISPR CAS9 system to the proper spot https://www.ozbiosciences.com/content/58-transfectionreagents-for-genome-editing 3’ 5’ CRISP-Cas9 • Once cut by Cas9, the specific DNA sequence undergoes repair using Non-Homologous EndJoint repair (NHEJ) • NHEJ will induce an insertion or a deletion (InDel) in the double strand break (DSB) that will result in a Stop codon producing a knockout Triggers endogenous DNA repair mechanism CRISP-Cas9 • Once cut by Cas9, the specific DNA sequence undergoes can also udergo repair using Homologous Recombination (HR) Downstream and upstream of cut site • HR being guided by homology sequences, it allow precise gene editing at the site of DNA cleavage • It is thus possible to introduce (i.e. to knock-in) specific point mutations to mimic a genetic dieases or modify specific aspects of the encoded protein • It is also possible to replace the endogenous gene by an entirely different genetic sequence (see next slide) in middle, insert sequence you wish to add (ex. Mutation) Knock-in CRISP-Cas9 Cat cut dsDNA in very specific way w/ CRISPR/Cas9 Keep reading frae in tact - do not disrupt gene sequence - allow for knock-in ex. Replaced nucleotides to produce point mutation some cells might effectively incorporate donor DNA in dsbreak Homologous combination to insert gene at specific site not perfect, need to screen for cells that have undergone proper repair and inseretered gene at right place Tissue-specific and/or inducible models • A major limitation of classic germline knockout mouse models is embryonic lethality in cases where the targeted gene is crucial for development. • Experimental strategies such as the Cre/lox system have thus been developed to allow : • specific deletion only in selected tissues and/or • induction of the knockout postnatally avoid problem of embryonic lethality • have full control of when Cuts DNA at specific sites that are recombinase can recognize - region of interest will be removed - end up w/ knockout Cre recombinase is a tyrosine recombinase enzyme derived from the P1 bacteriophage. The enzyme carries out site specific recombination event. Site-specific recombination occurs between two DNA recognition sites called LoxP sites These are 34 base pair (bp) sites consisting of two 13 bp palindromic sequences which flank an 8bp spacer region. Tissue-specific and/or inducible models Step 1 • Mice carrying a LoxP flanked gene on two alleles is generated using the transgenic embryonic stem cell injection method. Downstream and upstream of region you want to remove • These mice are commonly termed floxed mice for Flanked introduce LoxP in gene you want to knock out Lox • They are « Knock-out ready » gene is still normally expressed; these mice are still normal Step 2 • Mice expressing the cre recombinase gene is generated using the transgenic embryonic stem cell injection method Can generate mice that express it everywhere or… • Addition of various promoters upstream of Cre can allow helps control the expression of gene X tissue specific expression of Cre. • Ex: The Albumin promoter targets Cre expression to This is where albumin is made in our body; liver promotor is not expressed elsewhere • Ex: The Pax7 promoter targets Cre expression to muscle stem cells Tissue-specific and/or inducible models Step 3 • Cross the Cre mouse with the floxed mouse • Approximately 50% of the offspring will be heterozygous for the loxP allele and hemizygous for the cre transgene. some will carry albcre allele These animals are not knockout yet; they still have one gene that is intact Tissue-specific and/or inducible models Step 4 • Cross the hemizygous Cre heterozygous floxed with a homozygous floxed mouse • Approximately 25% of the progeny from this mating will be homozygous for the loxPflanked allele and hemizygous for the cre transgene. These will be your experimental mice. • About 25% of the offspring from this mating will be homozygous for the loxP flanked allele but will have no cre transgene. These will be your control mice *The remaining 50% of the progeny will be hemizygous cre heterozygous floxed that can be used for breeding Cre will recombine and remove critical region of gene X end up w/ full knockout of gene X in the liver • gene will not be knocked out in liver during early phase of development when liver is not yet fully formed and does not expresss albumin yet Tissue-specific and/or inducible models • In reality multiple controls are initially necessary to show that the observed phenotype is strictly due to deletion of the gene rather than to the presence of LoxP and/or Cre Controlling for the potential impact of having cre in mice Tissue-specific and/or inducible models Can combine models • ex. Can use tamoxifen to drive crerecombination whenever we want and cre expression is limited to cell that express Pax7 promotor, which is in muscle stem cells • can decide to remove Parkin or pink1 gene in muscle stem cells specifically when animals reach 10 weeks of age • have full control over the tissue we want to target/cell type and the timing when we perform this knockout • Inducible knock out models have also be developed such as the CreERT2 model • It utilizes a mutated estrogen receptor (ER) fused to Cre as a transgene (CreERT2), which only becomes activated and then translocates into the nucleus upon binding of the active tamoxifen (TAM) metabolite 4-hydroxytamoxifen (4-OHT). Edified promoter that allows are activity too eb turned off/on • modified receptor made specific to react to tamoxifen pups have cree and the two flexed alleles • cree inactivated at the begging since gene is fused to estrogen receptor — this keeps cre out of nucleus, where it can’t access LoxP • receptor move to nucleus and binds to regions on the genome, triggering gene expression Estrogen analogue Tamoxifen binds to estrogen receptor and triggers the movement of cre inside the nucleus • there, it will meet the Loxp site and cut and excise the gene Tissue-specific and/or inducible models • In this example, the Parkin gene was inactivated in the heart using the Myh6 MER-CRE-MER (Murine Estrogen Receptor) No effect on cardiac morphology if knocked out in young adult mouse • cardiac histology and function was normal • however, if you knockout Parkin at birth, animals would die by heart failure 21 days later • if you knockout Parkin at critical stages, it can be lethal In cardiac muscle cells cardiac specific promoter cre recombinase flanked by two modified MER • The cardiac specific promoter is that of myosin heavy isoform 6 Contractile proteins in the sarcomeres • LoxP site were flanking exon 7 of Parkin Loxp sites flanking exon 7 • will be excised and knock out Parkin expression Genotype mice to make sure they express floxed allele and have cre • tail clip, isolate DNA and perform PCR w/ primers to detect parkin gene and another to detect cre 600 bp band indicate the presence of the floxed allele (Park2 fl) 400 bp band indicate the presence of the normal allele (wt) Once it has been excised makes fragment smaller since you removed entire exon 297 bp indicate that the floxed allele was deleted (KO) • Note that the KO band only appears in mice that express CRE Mouse models have become broadly available All searchable databases North America Europe https://www.komp.org/ Embryo bank for genetically modified animals https://www.eummcr.org/ produces and characterizes 20,000 different gene knockout models • have repository and basic info on phenotype of these animals Mouse models have become broadly available • This international consortium has developped efficient and flexible tools to target and delete a vast number of genes Tens of thousands of different genes in the databases • They have also created large and diversified repositories of publically available mouse models for researchers Enter Gene ID Mouse models have become broadly available • Seach can be made using gene IDs Can also search thru key words of disease • Some genes are not targeted if: • It is targeted by another consortium (ex EUCOMM) • There has not been a strong interest by the community • Etc… Models generated depending on demand Search result for the mitochondrial peptidase LonP1 Product available in plaid vectors and ES cells w/ proper inserts • no live mice available Mouse models have become broadly available Name of gene • Product can be available under various forms depending on the stage of the particular KO project • Vectors • ES cells • Live mice • Cryo-recovery • Microinject • Sperm • Embryos most simple but not always available genetic info The knockout first strategy used by international consortia • The knock out first strategy has allowed efficient and flexible targeting of genes to knockout • Bacterial artificial chromosome (BAC)-based targeting vectors are inserted by homologous recombination into mouse ES cells. Knockout first strategy Knockout ready mouse Gene trap targeting cassette FRT equivalent of Loxp site • flank region you can remove when recognized by flipper enzyme Critical exon Loxp site flanks this Exxon • These vectors contain a gene trap targeting cassette containing a reporter tag (LacZ), a Neo cassette and LoxP sites flanking the critical exon to be deleted. • FRT sites are also flanking the LacZ tag and Neo cassette. FRT sites are recognized by the Flipase recombinase (Flp), an enzyme similar to Cre recombinase The knockout first strategy used by international consortia • Crossing the knockout-ready mouse (tm1a mouse) with a Cre expressing mouse to produce the tm1b mouse will remove the critical exon, resulting in a genetic KO. • Note that the LacZ tag is still present, which allows to vizualize in which tissue the gene of interest (both wild type and KO allele) is expressed Knockout first strategy Gene trap targeting cassette Critical exon Produces blue colour in organs where gene is normally expressed normally in the body Important info when you generate new knockout mouse model want to see where gene is highly expressed, at what stage of embryonic development does it start being expressed? can expect issues to arise to particular organs and not others if KO The knockout first strategy used by international consortia • Crossing the knockout-ready mouse (tm1a mouse) with a Flipper mouse to produce the tm1c mouse will excise the LacZ tag and Neo cassette Knockout first strategy Gene trap targeting cassette • Note that the critical exon will remain intact since it was not flanked by FRT sites. The tm1c mice are thus KO-ready but no longer express the reporter tag lacZ and neocassette have been removed • not knockouts yet since they still expresss exon 2 • Subsequent crossing with a Cre mouse to produce the tm1d mouse will result in a knockout. From tm1a, you can create different versions of a mouse to track gene expression during development to see where it is normally expressed, and once you know, you get rid of the lacZ gene and the blue colour and generate a pure knockout and start working Generate knockout Loop site shouldn’t be there anymore Where are these animals localized? Which database/repository? • J is Jack’s, biggest suppliers of lab mice globally White mouse Black mouse Nomenclature to codify genetically modified mouse donated genetically modded ES cells knockout first strategy Lets try to plan a knockout mouse project The idea to explore: You want to generate a mouse model to examine the role of PARK6, a gene involved in early onset familial forms of Parkinson’s. Using the available databases, propose a mouse model study the role of this gene in the main area of the brain affected in this disease. https://www.jax.org/search?q=Cre https://www.mousephenotype.org/ Search relevent Cre mouse lines https://www.uniprot.org/ https://www.ncbi.nlm.nih.gov Search relevent KO mouse lines Identify a protein of interest