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45 Cards in this Set
- Front
- Back
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Haploinsufficency
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recessive dominance from a loss of function of the other paired allele, which had initially been meeting a 50% product threshold
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3 consequences of recessive dominance
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haploinsufficiency
loss-of-function constituitive |
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in order to study genetic effects, can develop models by
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Developmental Mutants
Transgenics |
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Developmental Mutants
(5) |
Chemical: point mutation
Radiation: deletion Base addition/deletion: frame shift (usu chemical) Knockout: homologous recomb Transposon Insertion: esp Drosoph (transposase at random) |
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Loss of function can be varied
3 |
Complete loss
Allelic Series Haploinsufficiency |
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Gene Eliminating Techniques
2 |
Knockout: Homologous Recomb
**mouse only Gene Trap (if splice acceptor is ahead of gene) |
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Targeted Mutagenesis
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Homologous Recomb
(Can be knockout or insertion) |
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Loss of Gene Activity Techniques
5 |
Dominant Negative: protein truncation
Domain Swap: altering txt activity Antisense: Morpholino iRNA Neutralizing Ab Gene Trap (if inserted w/in gene) |
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Forward Genetics
using |
Starting w/ observable mutated phenotype,
in order to find the affected gene Use Positional Cloning via RFLP Microsatellites |
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Over/Expression Techniques
4 |
Positional Cloning: Promoter driven expression
Enhancer Trap Homogous Recomb Retrovirus |
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Dominant Negative Mutant
created by used for |
Used especially for things that need to dimerize like receptor mol (TGF-B)
non-functioning; interferes w/ wt, thus making other allele genetically dominant Freq are truncations by aa change ie. supersat of mRNA receptor mol microinjection to force improper dimerization |
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Domain Swap
Effectors? |
Used to deduce txt effector affects by swapping out the effector behind the binding site (not promoter)
the same effector will increase txt effect, and opp will create opp effect VP16: super-activator EnR: super-repressor |
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Gain of Function Mutant
Data Reliability? |
Constitutive
Genetically Dominant Data is not as reliable b/c these can be overexpressed and lead to Ectopic Expression Ie. txt non-responsive to regulation Ie. receptor truncation TGF-B binds Type II which then binds Type I Phosphatase Smads -> Smad Smad into nucleus |
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Maternal Effects
Maternal v. zygotic in early embryology Dependent On |
The embryo is loaded w/ mRNA and translational proteins to guide early development
txt machinery non-functioning Zygotic genome inactive at this stage Does not activate until inbetween cleavage and blastula Dependent on Maternal Health if Mother is Mutant, then the embryo can develop improperly |
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Mutation Rescue
pathway |
Inserting a missing gene product of a mutated gene to recover the effect
Typ used to confirm mutated gene a mutant will be formed if the product is upstream of ablated gene |
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Repression Genetic Pathway
Double Mutants |
look for gene that has only one type of outcome, that will be the last in the pathway
Look for the one that is different, that will be in the middle (look in info, not cross) The last one will be the first one and will resemble the last product |
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Epistasis
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When the an upstream gene products effects the activity of the downstream gene product
Activation Repression |
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TS strains
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Using Non/Permissive T
Non= usu lower T Typ creates a malformed protein- weak loss of function Can b used to take embryo past lethal point, then mutate effect Cyclops: w/o creates a cyclops Nodal: involv in cell differentiation |
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Autonomous
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Usu due to txt factor b/c it is independent, therefore intracellular
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Non-autonomous
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Due to Extracellular signals
Does not mean that the mutated gene codes for the loss of product (b/c dependent-extra) |
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How to check if the mutated gene is non/autonomous
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transplant wt into mutant
or transplant mutant into wt |
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Epigenetics
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Affecting genetic expression by modifying mechanism, and not due to underlying DNA cchanges
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Mosaics
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Organisms that express 2 different genotypes
In mammals: Barr body |
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Mosaic Behavior is not Mosaic
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Mosaic Behavior: corresponding anatomical change from resection of embryo
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Pole Cell Transplantation
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Transplantation of germ cell into a different host
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Reverse Genetics
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Starting w/ the gene and investigating all the phenotype changes
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Transgenesis Techniques in animal models
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Drosoph & C. elegans: transposon
Mouse and Zebrafish: microinjection into fertilized embryo Frog: DNA incorporated into sperm |
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Transgenesis Insertions
Placement Designed w/ |
Usually random
Homologous Recomb (targeted) Driven: behind the promoter Disrupted: w/in gene Rescued: anywhere in genome Often designed w/ insulation (DNA flanking) from other genomic effects |
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Transgenesis Embryo
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at the top of the trophoblast will be the Inner Cell mass, which contains the ESC
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Totipotent v Pluripotent
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Tot=Total
Pluri= Many, but not all |
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Chimera
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Created from two different genotypes from injection or blastocyte aggregation
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F1-3 transgenic
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F1 x wt = F2
F2 x F2 = F3 F3 will only show 25% mutant if homozygous recessive Therefore, this is done to incorporate into germline |
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How to maintain a homozygous lethal recessive
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Need to keep crossing w/ heterozygote-
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Balancer Chromosomes have
3 |
Lots of inversions so no allelic recomb
has recessive lethal (if two balancers-> die) genetic marker |
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Probing Forward Genetics
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Create mutant, ie. transposons
Find gene by amplification using polymorph probes- Positional Cloning RFLP Microsatellites: small repeats Southern: Run gel and attach probes Look for common allele from fragment size (and intensity) in observable mutant v wt Intensity is from homo allele; heteros will be paired light bands Check against known genes in area Check by rescue analysis Is it non/autonomous |
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Enhancer Trap
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In order to drive over/expression of inserted transgene
Insertion is random, b/c the cassette has a minimal promoter also has reporter marker used to create overexpressed strains quickly Chix: retrovirus But typically inserted in between promoter and gene Can b used to deduce whether non/autonomous |
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Gene Trap
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Used to disrupt function (protein truncation) or gene ablation
Insertion is random w/in intron Uses a splice acceptor, reporter, gene and Poly-A This fuses an exon w/ the gene, (cellular splice donor) which may lead to whole gene ablation |
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Knockout
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Mice only one that is able to be done using homologous recomb!!
Must use other tech w/ others this is targeted create cassette by cloning tech and use electropolration to fuse: transgenic procedure |
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Retrovirus Insertion
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After removing virulent portion
Using RT and integrase Must be applied to an infectious competent cell look to see whether it is replication in/competent |
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Replication In/Competent
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Incompetent: expression only affects cells that are infected
Competent: expression will spread to surrounding cells |
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Gene Inhibition Techniques
4 |
These are used b/c other models are unavailable to do KO
Dominant Negative (protein confirmation) Domain Swap (txt) Antisense (mRNA) Morpholinos iRNA Neutralizing Ab |
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Dominant Negative
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transgene or microinjection for gene disruption
non-functioning gene, but will affect proper folding of protein Used to "KO" other models |
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Domain Swap
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exchanges the txt effector
like will increased its activity (activation/repression) used to "KO" other models set behind DNA binding site |
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Antisense
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Morpholino: using oglionu- attached to designed homologous mRNA section. Designed to occupy mRNA, which will then lead to degradation
(Frog, Fish, Sea Urchins) iRNA: using anti-sense for dsRNA! degradation by Dicer -> RISC (Drosoph, C. elegans) These are used to "KO" other models by disrupting protein translation |
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Neutralizing Ab
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Using Ab to neutralize for opsonization
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