19.+20.+21. Mice genetics Flashcards
What is forward and reverse genetic analysis?
Forward genetics - phenotype driven:
random mutation -> phenotype -> gene identification -> interpretation of gene function => uncovers genetic basis of phenotype
Reverse genetics - gene driven:
gene identification -> targeted mutation -> phenotype -> interpretation of gene function => uncovers gene function by targeted mutations
What is gene trapping?
Gene trapping - an approach to study gene function using random insertional mutagenesis introducing a tag to identify (an intermediate between forward and reverse)
Random tag insertion -> gene identification -> phenotype -> interpretation of gene function
What are the three approaches for studying gene function in vivo?
- Forward genetics: random mutation -> phenotype
- Reverse genetics: trageted mutation -> phenotype
- Gene trapping: random tag insertion -> following gene in phenotype
What is an example of a conserved gene in species development?
Pax6 for eye development with conserved am. a. across species
Explain what is a balancer chromosome
Balancer chromosome: engineered chromosome which prevents recombination during meiosis ensuring specific genotypes - because genes are inverted
How are recessive mutations studied?
Ex: Heidelbrg screen in Drosophila - forward genetics: crossing a fly with balancer chromosome + random mutation -> crossing until mutated recessive allele is segregated homozygous genotype -> can study recessive phenotypes
Explain the Heidelberg discovery
Heidelberg discovery - Hedgehog (Hh) pathway: Hh mutations in flies cause forebrain defects: decreased sonic hedgehog -> no separation of forebrain
What are the possible chemical mutagens?
- ENU (ethylnitrosourea)
- EMS (ethyl methane sulphonate)
How are zebrafish large scale screens performed?
Random mutation -> crossing until affected genotype observed as phenotype
Why are mice a suitable model for studying mammalian developmental processes?
Mice are suitable model species because:
- mammalian
- small size
- diet + environment
- reproductive efficiency (non-seasonal breeding) - av litter size 10, gestation 20 days
- long term record + DNA resources + sequenced genome
- tolerant to inbreeding
- embryos and sperm can be cryopreserved
- ES cells
- publically accepted for experiments
What are the apparent differences between mouse and human genomes?
Mouse genome: 19 autosomes, telocentric chromosomes, 37k CpG islands
Human genome: 22 autosomes, metacentric and submetacentris chromosomes, 45k CpG islands
=> 70-90% sequence homology - suitable to be used as models for human processes - syntenic genes (almost complete synteny on X chromosome)
Explain the way in which ENU functions
ENU - supermutagen:
- transfers ethyl group to oxygen + nitrogen radicals in DNA -> mispairing
- ex: single base pair substitutions in spermatogonial cells at high efficiency
- each F1 generation may carry up to 100 mutations (if one locus mutated - can impact many phenotypes)
Missense vs nonsense mutation
Missense mutation: am. a. substitution
Nonsense mutation: premature protein termination - stop codom
Hypomorph vs antimoprh vs neomorph alelles
Hypomorph: mutant allele retains some gene function, less severe than LOF
Antimorph: mutant allele that antagonises normal gene function
Neomorph: mutant allele acquired new function
What are the genotypes of inbred and outbred mouse strains? What are their features?
Outbred:
- approximates human population
- heterozygous vigour
- maintain mutants as heterozygous - don’t die
Inbred:
- unique strain
- brother x sister >20 generations
- fixed genetic background - high probability of homozygosity - every individual is identical for autosome genotypes (X and Y diff)
-> mutations can be studied because background is fixed - other genes won’t interfere
Explain what is genetic linkage?
Genetic linkage: association of alleles which influences them to get transmitted to offspring in parental combination more frequently
How is recombination frequency influenced by distance between genes?
The further apart - the higher rate of recombination on the chromosome
Explain what is serial backcrossing?
Serial backcrossing - allows to determine which gene is responsible for a specific phenotype:
cross with inbred strain -> introduced new trait into neutral background -> cross offspring with parent again -> congenic strain created
What are the two commonly used DNA components used as markers?
Microsatellite markers - dinucleotide repeats (ex (CA)n where n=10-60) distributed in the genome - repeats are varying between mouse strains
SNPs - specific shared between strains but also can vary - can be used for identification
How can DNA sequences be identified that stay with the trait after multiple rounds of backcrossing?
Identifying DNA sequences that stayed with the allele for specific trait:
1. Design primers for PCR
2. Amplify by PCR
3. Sequence PCR products
4. Compare sequences from control vs trait affected
How can functional gene identification be performed?
Functional identification:
- backcrossing - gene region of the trait identify with markers (microsatellites / SNPs)
- gene region cut into diff segments - diff segments inserted into diff BACs
- diff BACs transformed into diff mice
- identify which BAC produced the target phenotype - BAC complementation
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What are the stages of early development?
Fertilization -> morula -> blastocyst -> embryo
What is are the processes that lead fertilised egg development into implantation stage embryo?
- Ovulation
- Fertilization
- Early cleavage
- Compaction
- Hatching
- Implantation
Explain what is pronuclear transgenesis?
Pronuclear transgenesis:
- Collect fertilized eggs from mother I
- Inject male nucleolus of fertilized egg with purified target sequence from BAC/YAC (fragment amplification) - DNA inserts randomly
- Transfer into mother II - pseudopregnant (mated with sterile males)
- Observe phenotype in born pups
What tool is used for pronuclear transgenesis?
Inverted microscope with 400x magnification with micromanipulator: right - injection pipette, left - negative pressure holding pipette
What are the potential effects of new gene introduction via pronuclear transgenesis?
- Position effect: integrates randomly - chromatin state determines if will get expressed / not expressed
- Mutagenic: integration may disrupt a gene - mutation
- Concatemers: injected DNA can recombine and form concatemers before integration (usually introduced as multicopy arrays)
- Not intact: injected DNA may be partially degraded - not all sequence integrated - transgene insertion not intact
What are concatemers?
Concatemers: a continuous DNA molecule that contains multiple copies of the same DNA sequence linked in series
Why is each strain of pronuclear transgenesis unique?
Each strain of pronuclear transgenesis is unique:
- irandom integration - diff integration sites
- different copy numbers (may be degraded/ recombined - change lengths)
- integrity of transgene may vary because not all will be functional - can insert into heterochromatin / be mutagenic
What are the two most commons reporters in developmental biology?
LacZ and GFP
How can chimeric organisms be produced?
Plasticity in early embryos - can combined cells from 2 diff embryos - form chimera - develop into chimeric animal
Explain the structure of the blastocyst
Blastocyst:
- epiblast -> fetus
- hypoblast -> yolk sac
- trophoblast -> placenta
ICM = epiblast + hypoblast
Which structure are ES cells derived from?
ES cells taken from epiblast (develops into fetus) - pluripotent - capacity to differentiate into all germ layers: ectoderm, endoderm, mesoderm
However, can ES cells can give rise to tumours - teratocarcinomas if injected into random locations (ex under skin)
The schematic of embryo development
How are ES cells derived and cultured in vitro?
ES derivation and culturing:
mice mated -> blastocysts taken out -> blastocysts cultured on feeder cells in vitro -> subculture by separating -> necessary signalling molecules to remain pluripotent / induce differentiation
How are chimeric animals made with ES cells?
- Blastocysts isolated from mice strain 1 -> ES cells from strain 2 injected into blastocysts
-
Implant chimeric blastocysts into pseudopregnant mice -> birth chimeric pups
=> but not all will be chimeric - not all transgenic because - not all ES will integrate
What are endogenous genes?
Endogenous gene: gene originating from within a living system - in contrast - exogenous - originate from outside (transgenic)
How are founder transgenic mice strains obtained?
Chimeric pups from foreign ES transgenesis are bread with the original strain type (the strain that provided the blastocysts - genetic background) -> bread until chimeric genotype isolated
How are ES cells genetically manipulated?
- DNA vector designed for HR with the target gene
- The vector can be integrated into ES cells by:
- Gene targeting: via HR - only integrates if homolog sequence found - HSV-tk not integrated
- Random integration: insertion into random location - HSV-tk integrated - will be screened and eliminated in the experiment - select against random integration
What are the two types of genetic vector integration into mouse genome?
Vector integration can be:
- Gene targeted integration
- Random integration
What are the parameters important for gene targeting in vector integration?
Parameters of vector for successful targeted integration:
- length of homology - the longer the higher freq of integration
- size of non-homologous sequence doesn’t make difference
- Isogenic DNA constructs increase integration freq -> using non-isogenic DNA causes mismatches
Explain what is isogenic and non-isogenic DNA
Isogenic DNA: sequences that come from genetically identical / nearly identical organisms (ex from inbreeding) - very similar / identical sequences
Non-isogenic DNA: sequences that come from genetically different organisms within species - between species
Explain the experimental approach of gene targeting
Gene targeting - specific integration of vector into ES cells
- Introduce vector into cells - adding selection markers - screen for cells based on introduced selector (ex ganciclovir)
- Replate resistant colonies - further culturing in well plates
- Identify inserted gene by Southern blot/ restriction enzymes + Southern blot
- Freeze other cells for later / use for chimera production
What experimental technique is used to screen for targeted clones?
Southern blot: probes for specific target sequence hybridisation - see if integrated into cell genome
Restriction enzymes: will identify change in cut endogenous fragment size if integration occurred - confirmation that the specific target sequence integrated - by Southern blot hybridisation
PCR: primer designed within transgenic target sequence + primer for the sequence from other side ->
if targeting successful, PCR will amplify the sequence using the primers and produce specific length products
if random integration - the products will be longer than from gene targeting
How can a specific gene expression pattern be investigated?
Inserting gene reporters into the target gene locus - LacZ / GFP - fused in frame to the upstream locus - integrated ino genome via HR
=> instead of exons 3, 4, 5, 6 - some parts deleted - the target vector inserted instead in recombination - could lead to problems if they are regulators / ORF -> target vectors can also be integrated without deletions
What is a knock-in (KI) vector?
Knock-in (KI) vector: a cDNA fragment integrated into genome to be expressed under control of an endogenouse gene - ex reporters (LacZ, GFP) - controlled by promoter of an existing developmental gene
What are multicistronic constructs?
Multicistronic constructs: genetic constructs/vectors that carry multiple genes within a single transcript - coding ORFs are linked and transcribed together as single mRNA - simultaneous expression of multiple proteins from one transcript
Consists of: shared regulatory elements, IRES, 2A peptide sequences
What is the structure of multicistronic constructs and what are the roles of each component?
- Shared regulatory elements: simultaneously controlled expression
- IRES: allows CAP independent translation mRNA into proteins
- 2A peptide sequences: regulates expression to be at equal levels of each cDNA
Explain homozygous gene targeting
Homozygous gene targeting - 2 ways:
- using same vector as gene targeting but using different selection markers for same allele
- using resistant casette - increased levels of the first selection marker gives both target alleles
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How are heterozygous / homozygous gene targeting products selected?
Most integrations random - gene targeted integrations in ES can be selected using drug selection - which don’t die replated - purified colony obtained - targeted genetic integration checked in DNA analysis (Southern) - ES lines used for pronuclear transgenesis - backcross to obtain transgenic strains -> intercross to obtain homozygotes / heterozygotes
How is gene targeting used for gene function analysis?
- Reporter incorporated into target gene
- Pronuclear transgenesis
- Observe reporter in offspring - lineage tracing + localization
What are KO phenotypes?
Knock-out phenotypes - gene function disrupted/altered - effects observed in offspring using reporters for that gene + others (to visualize structures that will be affected)
What is gene redundancy?
Gene redundancy: in gene KO other genes can take over and compensate for the knock-out function
How can experiments be modified if gene knock-outs don’t work because of gene redundancy?
In gene redudancy loss of gene function is compensated - instead of knock-out edit for gene overexpression - observe effects
What are the transgenesis applications in mice?
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What is the modern approach to transgenesis that ensures gene targeting?
Generation of ES cells by HR - a lot of work + time consuming -> CRISPR-Cas9 used
- Cas9 endonuclease
- guide RNA (gRNA)
- target sequence
- protospacer adjacent motif (PAM “NGG”)
How does CRISPR-Cas9 cut and what are the components?
Components:
- Cas9 endonuclease
- guide RNA (gRNA) - contains 20nt protospacer (target seq) - designed upstream of protospacer adjacent motif (PAM) - Cas9 recognises PAM - cuts both strands 3nt upstream of PAM
Protospacer adjacent motif (PAM “NGG”)
How is a sequence integrated into genome after CRISPR-Cas9 cut?
CRISPR-Cas9 cuts both strands 3nt upstream of PAM - cut repaired by:
- non-homologous end joining (NHEJ) - for gene knock-outs - unpredictable result
- homology directed recombination (HDR) - introduce specific mutations, reporter genes - precise insertion/modification - cell repairs using insert as template
- two gRNAs target sites chosen - will delete a larger genomic region
How can CRIPR-Cas9 be used for pronuclear transgenesis?
Cas9 and gRNA injected into male pronucleus - repair via HDR integrating target sequence - blastocysts transferred into pseudopregnant mother II
However, off target effects possible - Cas9/guideRNA cleavage not always precise - need to choose target sequences with minimal homology to other genes / regulatory sequences
What can be used to reduce off-targets effects of Cas9 and guideRNAs?
- Choose target sequences with minimal homology to other genes / regulatory sequences
- Mutant Cas9 nuclease (Cas9D10A) - cleaves only one strand -> using two gRNAs specific for each strand in the double strand cleavage + Cas9D10A generates an overhang -> reduces off-target integration
Unlikely that there is another site with 2 specific sequences that create exact overhangs - reducing the chance of ds breaks leading to incorrect integration
What is conditional gene knockout?
Conditional gene knockout - technique that introduces KO in a specific organ/ tissue/ cell at a specific time
To investigate diff gene expression properties controling:
- time of KO (ex for KO not to be lethal / to investigate gene role in developmental stage)
- specific cell (localization) KO
- permanent tag (reporter gene - track lineage)
- mutation (activate mutationn at given time/tissue)
How to induce a deletion in conditional genetics experiment?
Site-specific recombination (SSR)
In-vivo - inducing mutation (deletion):
- insert loxP sites by gene targeting to flank target sequence
- insert Cre-recombinase expressing transgene -> recombines the sequence flanked by loxP -> DNA excised
How to induce a deletion at a specific time in conditional genetics experiment?
If deletion required at specific stage in development or adult -> use inducable deletor
ex: Cre-ERT2 and tamoxifen: Cre-receptor construct Cre-ERT2: Cre fused to mutant estrogen receptor ERT2 - responds to synthetic analogue of estrogen - tamoxifen -> upon addition of tamoxifen Cre moves to the nucleus - causes recombintation -> gene excision (deletion)
Time when tamoxifen added - chosen freely
Exlpain the experiment how was lineage tracing of Lgr5 cells in intestine lining crypts investigated?
2 transgenes - LacZ and CreERT2/tamoxifen - allowed to compare expression in different time points of development - expression of LacZ induced by tamoxifen addition time point
Lgr5 expressing cells - stem cells - the stem cells also inserted with LacZ - when differentiated - no longer stem cells - can be traced by turning on LacZ reporter
- Tamoxifen addded day 1
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Tamoxifen added 6 months
-> previously Lgr5 expressing cells occupied the whole crypt
How can conditional genetics be used to activate and repress gene expression?
For conditional activation / repression: mutated Cas9 (dCas - ‘dead’) - dCas9 with fused activation /supression machinery - when activator/supressor added - induces activation / supression machinery action
What technique is used in animal cloning?
Somatic cell nuclear transfer (SCNT): metaphase II oocyte enucleated- adult differentiated cell nucleus transferred into enucleated oocyte - grown in vitro to blastocyst - transferred into mother -> offspring
However, very inefficient procedure - chromatin structure (epigenetic marks) in differentiated cells different to embryonic
Nuclear donor cells: fibroblasts, T lymphocytes
What are the application of somatic cell nuclear transfer?
- Reproductive cloning - generating genetically identical animals
- Therapeutic cloning - generating ES cells/ tissue / organs for regenerative medicine (genetically compatible organs - no rejection)
Compare two method used in generating ES cells?
Reprogramming by:
- IVF: oocyte fertilized with sperm -> blastocyst -> ES cells taken from epiblast => 99% survive to generate ES cells
-
SCNT: oocyte enucleated -> somatic nucleus injected -> ES cells from epiblast => 5% survive to generate ES cells
—> due to abnormal epigenetic regulation (chromatin structure in differentiated cells different compared to embryo) - inheritance independent of DNA sequence
Are maternal and paternal pronuclei different? What confirms it?
Yes, different
In pronuclear transfer - if paternal pronucleus X exchanged with maternal pronucleus - no embryo development => maternal and paternal are different pronuclei - both required for normal development => gene imprinting in gametes
What is parental gene imprinting?
Parental gene imprinting: some genes activated / repressed depending on parental origin -> monoallelic uniparental expression
Imprinting occurs in gametogenesis exclusively in eggs / sperm -> different imprinting marks -> persist into the zygote
Both maternal and paternal needed because gene expression must be complementary
Explain how sex specific genomic imprinting occurs
- Supression/activation erasure in primordial germ cells
- Imprint establishment depending on embryo sex (birth-> puberty)
- In reproduction both gametes with different imprints form zygote -> blastocyst
- Monoallelic expression but genetic imprinting compatibility between males and females - expression of needed genes ensures normal development
How are the imprinted genes organised?
Maternally/paternally imprinted genes are usually clusterred in chromosome regions - imprinting controlled by same mechanisms in the region
How are imprinted genes studied?
Imprinted genes can be revealed by mutation: mutating gene which supposed ot be expressed maternally/paternally -> gene not functional in both (full mutation)
Studying a gene on paternal X:
if abnormal phenotype => mutated gene is paternally expressed
if normal phenotype => mutated gene is maternally expressed
What are the major roles of maternally/paternally expressed genes and what is the main mechanism of imprinting?
~80 imprinted genes identified - expected more - have major effects on fetal/placenta growth, postnatal behaviour
Maternally expressed genes - supress fetal growth
Paternally expressed genes - enhance fetal growth
DNA methylation - main mechanism but also epigenetic modifications also used
How differentiated cells can be reprogrammed into pluripotent cells?
iPSCs:
24 TFs identified to reverse differentiation - only 4 TFs needed for in-vitro de-differentiation: Oct4, Sox2, Klf4, c-Myc sufficient to turn mouse fibroblasts into iPSCs - use retroviruses to infect embryonic fibroblasts with the needed de-differentiation genes - re-differentiation using specific signalling molecules
What is the potential of iPSCs for therapeutic use?
iPSCs can be generated from patient’s cells and re-differentiated into needed cell type - genetically compatible no rejection
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