BC6 Flashcards
What are the advantages of yeast as a model organism for the fundamental processes in eukaryotic cells?
- Cell cyle (incl. DNA replication, mitosis)
- Protein life cycle (transcription, translation, folding, sorting, degredation)
- Organelle structure and function
- cellular stress response (DNA/protein)
What are the limitation of yeast as a eukaryotic model organism?
- Small (less well suited for microscopy techniques)
- Contains cell wall (certain chemicals not taken up)
- No complex development processes (unicellular)
What are the two types of yeast in the lab, and what is a defining difference between them?
- Budding yeast (Saccharomyces cerevisiae)
- Daughter cell is produced through budding
- Fission yeast (Schizosaccharomyces pombe)
- Cell division occurs centrally
What are the kingdom and phylum of yeast?
Kingdom: Fungi
Phylum: Ascomycota
How long ago was S. cerevisiae and S. pombe common ancestor? With which animal is this a similar evolutionary distance?
- 109 years ago
- Humans
Why is the long evolutionary distance between the two yeasts and humans relevant to model organisms?
Because if a mechanism is conserved between the two yeasts, you will most likely find it in humans as well!
What are the advantages of S. pombe as a model organism over S. cerevisiae? Name 4 examples.
Certain aspects of cell biology are more similar to humans, such as:
- Splicing
- RNAi
- replication origins
- Centromeres
What are 2 advantages of S. cerevisiae as a model organism over S. pombe?
- More widely studied
- More experimental tools
What attributes make yeast a superior genetic model organism compared to other eukaryotes? What are its limitations?
Advantages:
- Cell cycle (includes DNA replication, mitosis)
- Protein life cycle (transcription, translation, folding, sorting, degredation)
- organelle structure and function
- cellular stress response (DNA/protein)
- grows quickly
- grows on solid and liquid media
- cultivation is simple and inexpensive
- genome small with simple structure
- high recombination rates
- high transformation rates
- haploid life cycle allows direct genotype phenotype relation
- crossings are easy
Disadvantages
- Small (less well suited for microscopy techniques)
- contains cell wall (certain chemicals not taken up)
- No complex development processes (unicellular)
What advantages arise from the haploid/diploid life cycle? How are crossings analyzed?
- genetic modification of 1 copy of gene is sufficient to investigate phenotype in haploids
- can use diploids for purpose of crossing two mutants or to propogate a heterozygote mutant under conditions where it does not display a phenotype.
- conjugation (mating) and meiosis (sporulation) can be easily induced experimentally.
Crossing analysis:
- tetrad analysis
- plate spores seperatley to test markers
- can also detect mating type by looking at mating type pheromone
What basic types of genetic interactions exist between two double mutants? How are these interactions interpreted?
- Additive/synergistic: different pathways
- epistatic: same pathway
- suppressive: two genes work in different pathways that negatively impact each other, or the suppressor lies upstream in the same pathway, and if absent the downstream part is less important.
How does the two-hybrid system work?
- basic two hybrid system (not a variation)
- have DNA binding domain (BD) attached to bait protein (X)
- have Activation domain (AD) attached to prey protein (Y)
- BD is bound to DNA upstream of reporter gene.
- If protein X and Protein Y, AD will get into proximity of BD, completing the transcription factor, and allowing transcription of the reporter gene.
Give the proper names for the following for your favorite gene 1:
- the gene
- the protein
- the mutant gene
- knock out/deletion
- a conditional allele
- YFG1 = the gene (your favorite gene 1) - italics
- Yfg1 = the protein - no italics
- yfg1 = the mutant protein - italics
- yfg1Δ = knock out/deletion - italics
- yfg1-1 = a conditional allele, more precise: yfg1=L64A
What is is the meaning in the budding yeast systematic name of:
YDR135w
Yeast (Y) - chromosome IV (D) - right arm (R) - 135th ORG (135) - Watson strand (w)
What are the following characteristics of S. cerevisiae?
- Shape
- size (haploid and diploid)
- Genome size (bp and chromosomes)
- How many genes and how many contain introns?
- usual chromosome type
- Tolerances
- Cell wall or not?
- Ovall shaped (with bud)
- 4 um (haploid), 5-6 um (diploid)
- 12 Mbp on 16 chromosomes
- 6400 genes, 4% contain introns
- usually diploid, but can also live as haploid
- wide range of tolerances
- 2.8-8.0 pH
- <3 M glucose
- <20% EtOH
- Has a cell wall
What are the following characteristics of S. pombe?
- Shape
- Size
- How many base pairs in genome on how many chromosomes?
- How many genes, and how many introns?
- Usual chromosome type
- other differences to S. cerevisiae
- Rod shaped
- size: 13x 3um (haploid), or 22x4 um (diploid)
- 13 Mbp on 3 chromosomes
- 5000 genes, 5000 introns
- usually haploid, but can have diploid cells which usually proceed directly to meiosis.
- has a longer G2 phase than S. cerevisiae.
Name the 3 types of allele and brief description
- Deletion allele: cpmplete lack of a gene
- Conditional allele: mutation that allows activation or deletion under specific conditions (eg. degron)
- Point mutation allele: mutation of one or several codons. May retain protein but induces a specific defect.
Name 4 cellular phenotypes
- growth
- survival
- subcellular localization
- cellular function (eg. DNA replication/splicing)
name 3 molecular phenotypes
- protein-protein interaction
- protein activity
- post translation modifications (PTM)
Draw a diagram depicting the budding yeast life cycle
Describe crossing
Done to generate a double mutant yeast strain. Basically crossing 2 single mutation strains to get a double mutation strain.
Describe conjugation/mating
spontaneous when both haploid strains are mixed.
In case of two strains, each with marker, diploid produced would be heterozygous for both markers.
describe Sporulation
Diploids will be grown and plated on sporulation medium.
Spores germinate, each giving rise to a colony.
Spore is haploid, essentially containing a chromatid, meaning that each colony is expressing a single chromatid.
List 6 genome wide approaches to genome analysis in yeast
- Gene expression: Micro-arrays (gene chip)
- Gene knockouts (barcoded deletion library)
- Protein overexpression
- protein localization (GFP)
- Protein-protein interaction (two-hybrid, affinity tag)
- protein purification
Define functional genomics
- Genome wide
- uses parts of genomics that are “dynamic” such as:
- transcription
- translation
- protein-protein interactions
- describes gene functionality
Define Systems biology
- uses holistic approach (as in looking at whole system together)
- tries to model and discover emerging properties of cells functioning as a system together
- requires quantitative data
Give a basic explanation or draw a diagram depicting DNA microarrays. What is the purpose of them?
Purpose: To determine RNA transcribed by a genome.
Classic experiment for:
- looking at expression
- quantifying mRNA levels
steps:
- mRNA is extracted from both control and experimental sample.
- experimental sample has been modified on some way (hypothetically affecting the RNA expressed)
- reverse transcription and fluorescent labeling of transcribed RNA generates tagged cDNA.
- cDNA from experimental and control pooled so there is an equal amount of both.
- passed over microarray with known oligo-nucleotides in specific, known places.
- can tell how much has bound to specific types of DNA by looked at fluorescence in known spot.
- cDNA from both control and experimental can bind to the same spot on array, creating a yellow color (assuming that control is green and exp. is red).
- if a spot is more red than green, experimental expressed more of the RNA that formed that cDNA than the control and vice versa.
What are 4 advantages of RNA sequencing over microarrays?
- High dynamic range (can measure lowly and highly expressed genes)
- Sensitive
- does not need reference genome
- splice variants
What is a yeast deletion library?
- array of yeast knockout strains
- each yeast strain harbors knock out of one particular gene
- robotics needed for screening
What is barcode technology (in the context of yeast genetics)?
- Each knock out strain marked with a unique “barcode”
- this is a unique sequence to that strain of knockout yeast
- barcode flanked by common sequence used in PCR amplification
- DNA analyzed by microarray or next gen sequencing
What is a competative growth assay?
- Barcodes used to identify mutants
- grow pooled deletion mutants in presence or absense of drug (or other thing that might affect growth)
- barcodes are amplified by PCR
- next-gen sequencing/array used to identify which strains of yeast survived (via barcode)
What is a synthetic gene array?
- Cross a mutant from knockout collection with a mutant of interest, resulting in either limited growth or no growth (synthetic lethality)
- basically mate one mutant with all 4800 strains in library
- allow to sporulate
- take MATa haploids
- screen for double mutant and see which ones survive
What is a separation of function mutation?
- mutation that confers the loss of a single biochemical property.
What is an Emap of genetic interactions?
- A process for quantifying genetic data
- essentially creat an interaction score of each gene pairing
- each screen is one data set
- axes are the genes that are crossed
- color scale from aggrevating to alleviating
- allows resolving between specific and shared subunits of SWR-C complex.
- Example:
- yeast mutant gives similar emap compared to treatment with DNA damaging agent
- mutant causes DNA damage potentially
What is a protein Chip?
- Similar to microarrays, more technically challanging
- to see protein interactions
- biochemical properties of proteins more diverse than nucleic acids.
List 4 methods of genome wide protein interaction studies.
- Protein chips
- yeast two hybrid
- Affinity purification (quantitative proteomics)
- SILAC (metabolic labeling - quantitative proteomics) (mass spec used)
Did the “yeast genome project” lead to new experimental strategies? Which?
- Micro-arrays - gene chip
- Gene knockouts (barcoded deletion lubrary)
- Protein overexpression
- Protein localization
- Protein-protein interaction (two hybrid, affinity tags)
- Protein purificaition
How is an SGA experiment designed and carried out? How a competative growth experiment?
- SGA experiment (synthetic gene array)
- Cross a knockout mutant from gene library with mutant of interest and see if this causes synthetic lethality.
- Can cross 1 strain with 4800 yeast simultanously
- Competative growth:
- Barcoded samples, grow mutants under different conditions.
- See which barcoded samples from each grew better
Why are quantitative measurements a fundamental requirement of the E-map approach?
Computational analysis is needed to compare them, and it is based on a scale of aggrevating to alleviating, thus, it must be quantifiable to be able to compare so many.
Need to do an actual calculation to determine interation score, thus you need to start with actual numbers.
What are advantages and disadvantages of different genome-wide protein-protein interaction techniques?
- Protein chips
- Advantages: analogous to microarrays, so easy to read, and can look at a lot
- Disadvatnages; technically challenging, biochemical properties of proteins more diverse compared to nucleic acids
- Two hybrid
- Advantages: allows easy crossing and test of interactions with multiple bait strains.
- Disadvantages: not every protein is suitable (membrane associated, toxic); Many false negatives (differences in procedure)
- Quantitative proteomics:
- Affinity purification and metabolic labeling
Draw the lifecycle of Drosophila melanogaster
Draw a diagram of a fly breeding/crossing scheme
What would a dominant temperature senstaive mutation do?
Would cause fly not to be able to live at a certain temperature, like 29 C
What are balancer chromosomes?
- Can be used asd genetic tool to prevent recombination between homologous chromosomes
- allow populations of flies to be maintained without contstantly screening for mutations
- Contain multiple inversions, thus prevent recombination
- carry recessive lethal mutation, are homozygously lethal
- carry dominant marke
What are Heidelburg screens?
- experiments addressing cell fate and cell form
- Indicated that temporal and spatial pattern of zygotically active gene transcription provided triggers for controlling normal sequence of embryonic development
- established these genes:
- GAP
- pair-rule
- segment polarity
What are gap gene?
- Mutation results in loss of contiguous body segment
- resulting in missing that part
- basically cause deletions of particular segment
What are pair rule genes?
- result of differing concentration of gap gene proteins
- defined by mutation that causes loss of normal development pattern in alternating segments
What are segment polarity genes?
- Examples include hedgehog and wnt
- mutation leads to segments not being properly separated
How would one identify mutated genes in a genome with forward genetic screening?
- find genomic region by recombination complementation against deficiency collection
- identify correct gene by:
- comparing sequence of WT and mutant alleles
- genomic rescue experiments
How are genes characterized in forward genetic screens?
- describe the molecular features
- expression pattern
- type of protein
- analyze interaction with other genes
- genetic epistasis
- transcription/translational regulation
what are maternally required genes?
- 30 genes required in mother for normal embryonic patterning
- 3 organizing systems
- anterior
- posterior
- terminal
How do BCD and Nanos control expression of other factors?
- both are point source diffused
- Bcd inhibits Cad translation
- Nanos inhibits Hb translation
- is locational, so basically, Cad protein will not be in the same location as Bcd protein, and the same for Hb and Nanos.
Give examples of ubiquitous expression in flies
- Cad and Hb are examples of ubiquitous expressin
- so basically their RNA levels remain constant everywhere in cell, as opposed to point source diffusion genes, where RNA is localized to one space
- the translation of Cad and Hb is regulated by Bcd and Nanos respectively
What are 2 examples of things that maternal factors and gradients can act as?
- Transcriptional activators
- Hb is an enhancer
- Morphogens
What is genomic rescue and what is it used for?
- Basically inject into the mutated organism a wild type copy of the gene you think is causative of the phenotype and check if there is a rescue
Draw diagram of the segmentation gene network hierarchy.
What are most genes in it?
How is it (mostly) regulated?
don’t need to include fly drawings, are just helpful
- Most genes are transcription factors
- regulation is almost entirely transcription factors, and is highly conserved.
What are cis-regulatory elements?
- Regions of non coding DNA that regulate the transcription of neighboring genes
- contain multiple binding sites for various transcription factors
define Modular organization
Expression domains generated by seperable cis-regulatory elements
What does autonomously active mean?
A gene is active independantly of basal promotor/genomic insertion site
What are 3 experimental approaches to determining transcription factor binding preferences?
- DNase footprinting
- in vitro measurements
- Chip-chip or ChIP-seq
What can construciton of a positional weight matrix do?
- Align experimentally determined binding sites
- calculate frequency with which each base occurs at a position
- for any given sequence, one can now calculate the strengths of binding based on how the sequence matches the PWM
Draw diagram depicting how stripes are made, what is this two step model based on?
Based on
- molecular epistasis
- expressin dynamics
- organization of cis-regulation
Define a morphogen
- is a molecule (transcription factor, ligand, receptor) that shows a concentration gradient
- depending on its concentration, the exposed cells or nuclei will undergo different fates
- Example:
- modification of Bcd copy #
- modification of number/strength of BCD binding sites
- Example:
What is an imaginal disc?
- one of the parts of a holometabolous insect larvae
- will become a portion of the outside of the adult insect during the pupal transformation
- contained within the body of the larva, there are pairs of discs that will form, for instance, the wings, legs, antennae, or other structures in the adult
Define a master regulator and give 3 imaginal disc master regulators?
- A master regulator is a protein (e.g. transcription factor) at the top of a gene regulatory hierarchy
- particulary in pathways related to cell fate and differentiation
- is both necessesary and sufficient for development of that organ or tissue
Examples
- Eyeless - late embryo, early eye disc
- scalloped - late embryo and wing disc
- distal-less - late embryo and leg disc
What is the morphogenic furrow?
- contracted band of cells behind which photoreceptors differentiate
- wave of differentiation sweeps from anterior to posterior eye disc
- cone and pigment cells differentiate during pupal stage
composition of compound eye of drosophila
called the ommatidia, composed of:
- cornea - transparent outer region
- retina - cellular layer
- 8 photoreceptor cells (R1-8)
- 4 cone cells
- 2 primary pigment cells
Draw the eye disc fate cascade
What is a molecular epistasis experiment, what is an example of what it can help determine?
- used to determine the order of action of genes in a regulatory hierarchy
- phenotype of double mutant is compared to that of a single mutant (or wt vs mutant)
- Can help to determine components of drosophila eye disc fate cascade.
Define “sufficient” and “Necessary” in the context of imaginal disc
- Sufficient:
- overexpression of gene induces additional formation of that structure in different body parts
- Example:
- overexpression of toy induces formation of eyes in different body parts
- Necessary:
- lack of gene causes structure to not be there
- example:
- lac of eyeless causes the absence of the eye
What is the UAS/GAL4 system and when might it be used?
- used to study gene expression
- by crossing one fly with GAL4, and one with UAS, the fly resulting with both of them will allow gene with UAS to activate the GFP (or any other gene, such as toy) transcription since GAL4 binding to UAS activates it.
Which 3 signalling pathways are important for the proper propogation of the flye eye?
Draw diagram showing them
- Hedgehog (hh)
- DPP
- Ligand: wingless (wg)
What is the role of Hedgehog?
- required for initiation of differentiation
- temp. sensitive allele
- hh signalling sufficient for photoreceptor differentiation
- acts redundantly with DPP in morphogenic furrow progression
- if no hh present: Ci converted to repressor
- hh present: Ci acts as gene activator
What is the role of DPP?
- essential for furrow intiation
- not essential for furrow progression
- is only sufficient to initiate differentiation at the anterior edge
- hh and dpp act redundantly in furrow progression
- molecular mechanism:
- binds to 2 receptors, bringing them close together
- receptors activate MAD protein
- MAD is transcription factor
- activates genes
What is the role of wingless?
- blocks morphogenic furrow movement
- it is necessary for furrow to stop moving at some point
- but not too soon
- Absence of wg: genes repressed
- Presence of wg: B-catenin transcription factor made, gene activated
What is mitotic recombination? What is it used for?
- Used to study role of dpp, hh, eg in eye formation
- Temperature sensitive alleles difficult to make, so use mitotic recombination
- Is followed by mosaic analysis
What is genetic mosaicism?
- When similar cell types in an organism express different phenotypes due to dissimilar genotypes at a specific locus.
Describe sequence of photoreceptor development in drosophila eye.
- R8 is founder cell
- followed by R25, R34, R16, R7
- other cells of ommatidia are recruited in the cluster by signalling pathways
What is atonal?
- is expressed in all cells just anterior to the furrow
- as furrow progresses, most cells that express atonal lose that capacity
- atonal expression behind the furrow is confined to the R8 progenitor through a process called lateral inhibition
Describe lateral inhibition
- feedback regulatory loop translates small differences in Ato gene activity into R8 vs non-R8 cell fates
- cycle of reinforcement: one cell will become the only ato expressing cell
- Atonal expression is refined by lateral inhibition until atonal is expressed in only the founding R8 precurser
- essentially, signal next to R8 cell prevent cells next to it from becoming neuronal precursers
By which pathway are the other ommatidial cells recruited?
The spitz-EGF receptor pathway recruits:
- photoreceptors R1-R7
- Cone cells
- pigment cells
What is a temperature sensative allele and what is it used for?
- Often cause loss of function at non-permissive temperatures
- allow researchers to induce mutant phenotype in mutants at non-permissive temperatures
- in order to study effect of messing with gene
- but allows organism to remain unmutated at permissive temperatures
What are inbred strains in mice?
- sister x brother matings for > 20 consecutive generations
- are almost all identical genetically (besides gender)
- homozygous for basically all loci
- one inbred strain will have a characteristic setting it apart from other strains
- most traits done differ between generations
- other traits can be manipulated with diet and environemnt
Name 4 mating systems and their applications
Draw a diagram depicting hormonal control of spermatogenesis
- Pituitary hormone effects:
- LH and FSH stimulate spermatogenesis and testosterone secretion by testes
- Testes hormone effects:
- Testosterone and inhibin inhibit the secretion of GnRH by hypothalamus
- Inhibit LH and FSH by pituitary
Draw a diagram depicting hormonal oogenesis
- pituitary hormone effects:
- LH and FSH stimulate oogenesis and hormone secretion by the ovaries
- Ovaries hormone effects:
- estradiol and progesterone inhibit the secretion of GnRH (gonadotropin releasing hormone) by the hypothalamus and LH and FSH by the pituitary.
Name 5 main steps of mammalian fertilization
- sperm binds to zona pelucida of egg
- acrosome reaction
- entry through zona pelucida
- fusion of plasma membranes
- entry of sperm nucleus into ovum cytoplasm.
What are the roles of Izumo1 and Juno during mammalian fertilization?
- During fertilization, a single sperm binds to the egg’s membrane.
- The protein Izumo1, which is tethered to the membrane of sperm, forms an adhesion complex with its receptor protein, Juno, which spans the egg’s membrane.
- Fertilization does not take place in the absence of this complex.
- After fertilization, Juno is lost from the egg’s membrane, thereby preventing the binding and fusion of additional sperm (to block polyspermy).
Describe the transcriptome changes seen during early development.
- ZGA = zygotic genome activation
- MAG = mid-preimplantation gene activation
- The time of major ZGA in different species is different
- mouse: two cell stage
- Human, pig: 4-8 cell stage
- Cow: 8-16 cell stage
- Major ZGA is the point at which the zygotic genome becomes activated
- This is part of the maternal to zygotic transition
- essentially, the transcriptome of the mom not being responsible for the zygote anymore
- at this point, the zygote’s transcription machinery takes over and does its thing.
Diagram for help visualizing, don’t need to memorize
How was transcription tracked in cows?
UTP uptake experiment
- Radioactive UTP was in cows
- UTP uptake was tracked with radiation
- UTP is needed in transcription, thus an increase in UTP uptake indicates an increase in transcription
What was cow fine mapping used for, and why were 2 species of cows crossed to do it?
- Crossed 2 species of cows because:
- were relatively distant relatives
- easier to differentiate between parental genomes since there would be more SNPs (small nucleotide polymorphisms)
- RNA extraction done at all stages from GV oocyte, to blastocyst
- in oocytes, mRNAs are all already spliced (by mom)
- when embyronic transcription starts, can see increase in introns
- these would have been spliced out in maternal mRNA already, so any introns would be from offspring transcription
- large increase in introns indicates point of transition from maternal to zygotic transcription
Name 3 strategies to detect de novo transcribed genes why would you need to do this?
strategies:
- emergence of new transcripts after fertilization
- e.g. transcripts detected in 4-cell embryos, but not in oocytes
- basically, transcript present in embryo, but not original egg
- indicates that it has been generated in embryo
- Detection of transcripts with breed specfic paternal SNPs after fertilization
- so if it is an SNP that is specific to the breed of the father, it wouldn’t come from the oocyte, since that would just be mothers DNA
- Appearance of unspliced transcipts (intronic reads), occurring during active transport
- Unspliced means it was transcribed in the zygote, since all maternal transcripts would have been spliced already
Why need:
- to determine at which point in the stages of early mammalian development is the maternal to zygote transition and the major zygotic genome activation
- ie. 2 cell, 4 cell, etc.
- 8 cells in cows
Which is the main receptor involved in embryo maternal cross talk?
- Answer: ErbB receptors
- Heparin binding EGF-like growth factor (HB-EGF) expressed in endometrium
- HB-EGF interacts with ErbB receptors on blastocysts
Name 4 techniques involving homologous recombination or nonhomologous recombination for genetic modification in mouse embryos.
Homologous recombination
- Enucleation and nuclear transfer using transfected donor cells.
- aggregation with/injection of transfected embryonic stem cells
Nonhomologous recombination
- DNA-microinjection into pronuclei of zygotes
- Viral vectors for gene transfer (retroviral, adenoviral)
Define forward and reverse genetic studies
- Forward genetic screen
- see phenotype
- try to figure out what gene is doing that
- alteration of genes
- determine gene function
- Reverse genetic screen
- see genotype
- alter gene
- see consequences
- determine gene function
Describe the lentivirus vector system
- lentivirus is genus of retrovirus
- can be used as a transfection vector
- 2 parts are made:
- lentiviral vector
- packaging constructs
- injected into packaging cell to create virons
- they burst out, and then they infect other cells with desired transgene
- dont have ability to replicate after this, preventing virus from bursting cells and killing them
Describe 4 types of stem cells
- totipotent
- most versitile
- sperm + egg form one fertilized egg = totipotent
- can give rise to any and all cell types
- can give rise to functional new organism
- first few divisions totipotent
- pluripotent
- can give rise to all cell types
- can’t give rise to new organism
- multipotent
- less plastic, more differentiated
- can give rise to limited range of cells within tissue type
- example: multipotent blood cells can turn into:
- red blood cells
- white blood cells
- platelets
- Adult stem cells
- multipotent stem cell in adult
- used to replace cells that have died or lost function
- undifferentiated in differentiated tissue
- renews itself
- can specialize to yield all cell types present in its original tissue
Which genes govern early mouse development?
- Cdx2
- turns precurser to Trophectoderm (multipotent)
- inhibits Oct4
- inhibited by Oct4
- Oct4
- Turns precurser to inner cell mass (pluripotent)
- inhibits Cdx2
- inhibited by Cdx2
- Nanog
- Turns inner cell mass to Epiblast (Pluripotent)
- inhibits Gata6
- inhibited by Gata6
- Gata6
- turns inner cell mass to primitive endoderm (multipotent)
- inhibited by nanog
- inhibits nanog
How does gene targeting by positive negative selection work?
- Thymidine kinase (TK) catalyzes reaction that converts ATP to TMP + ADP
- TK is necessary in creating thymidine
- TK is outside homologous regions
- if homologous recombination occurs successfully, TK will be outside target gene (positive selection)
- if TK is inside of target gene, it means that it was inserted into a random gene (negative-selection)
Describe a method of going from mutant ES cells to mutant mouse cells
- Inject blastocyst with embroniuc stem cells (ES cells)
- inject embryo into mouse
- chimeras are born
- mate chimeras
- get mix of wild type and heterozygous mutants
- breed heterozygous mutants together to create homozygous mutant.
What is the difference between a mosaic organism and a chimera?
- Mosaic:
- Presence of two or more populations of cells with different genotypes in one individual who has developed from fertilized egg.
- Chimera:
- Single organism composed of cells from different zygotes.
- prodeced by merger of multiple fertilized eggs
Describe the Cre/Lox system for conditional mutagenesis
- Similar to Flp/Frt system with drosophila
- used to create mutant mice (mosaic)
- Cre-recombinase is temperature sensitive, so it only works in a certain temperature range
- first generation of mice:
- cre mouse
- promotor then
- cre gene, then
- stop codon
- LoxP mouse
- promotor then
- loxP gene then,
- Target gene then,
- stop codon, then,
- loxP gene then,
- eGFP gene then,
- stop codon
- cre mouse
- Breed them together to create Cre LoxP mouse
- Cells with active Cre recombinase (at correct temp)
- will pull LoxP sites together
- the target gene is disrupted
- the 1st stop codon is not read, allowing expression of the eGFP gene
- Cells without active Cre recombinase
- original gene function untouched, and eGFP is not expressed
- Cells with active Cre recombinase (at correct temp)
