Lecture #8 - Mouse Genetics 2 Flashcards
International Knockout Mouse Consortium (IKMC) - Overall
Intiated in 2006 – intrenational group of researchers generated a resource for the community
Aim – Generate C57BL/6 mouse embryonic stem cells with a targeted KO or conditional KO mutation for every gene in the mouse genome (KO all 20,000 known genes in mouse genome)
- EXCPETION – won’t KO the gene if the mouse has already been generated
What does IKMC target
Target 20,000 known and predicted genes (target entire mouse genome)
They are trying to KO all 20,000 known genes in mouse genome
Strains used for KO/transgenics
Historically use 129 for KO/transgenics
- used because more germline compenent (compared to Black 6 which is harder to work with)
NOW have new line of Black 6 (Black6N) that have mutations that make it more germline competent
- Used more now because they are more similar to mouse commonly used in the lab
Use of IKMC
Use IKMC – instead of designing and generating mice yourself you can get the mice from these international conserciums
Can get the mice in the form of live mice or germ plasm
Common first step in experiment – look at IKMC to se if that have a mouse that is useful AND if they have phenotyping data
International mouse phenotyping consortium
Overall – phenotying mice
Aim - Generate C57BL/6 mice from mESCs with targeted KO or conditional KO mutations for every gene in the mouse genome
- GOAL – trying to understand what the phenotypic output of some genes are (for all 20,000 mouse genes)
International mouse phenotyping consortium - Mouse phenotyoing Process
When phenotyoing the mouse there is no starting hypothesis –> Means there is a set number of asays to try and understand some of the basic phenotypes of the mice
Used 1000s of conrtols and 9 KO for each genotype
International mouse phenotyping consortium - Data
Data from IMPC is very good
Can go to website and see if the gene that are are interested in has a phenotype
International mouse phenotyping consortium - Use
Use - Wanted to look to see if some genes of interst have a phenotype of interst
- Can tell you if there is something intersting that you should follow up with
Useful to see if gene of interest is embryonic lethal OR if they have a severe phenotype that it would be impractical to look at a KO (INSTEAD might want to make a conditional KO)
Good place to start when have an unknown gene
International mouse phenotyping consortium - KO
Doing a whole body KO
Because whole body KO –> if KO a lethal gene then the data will tell you if this is lethal
International mouse phenotyping consortium Example
Example #1 – gene Ccdc120 was found in an RNA sequencing screen –> once found they looked at IMPC to see if the data associates with the LOF of the gene
Example Phenotypes in International mouse phenotyping consortium
Test that are done on the mice:
1. Skull – whether the mice survive –> know if the gene is required for development (if the KO is avaible at weening) OR is there embryonic development
2. Startle reponse test
3. Blood glucose
4. Cardio Measures
5. Body weight
How does IKMC KO genes - Overall
Have a construct that they use to KO every gene
- Can be done when have unknown genes (NOTE - use the construct when have unknown gene ; IF you know things about the gene you might be more specific about which exon you remove)
- Vector gets incorporated into every gene in the mouse genome
Goal – Want to set up screen where you KO every gene in the genome (everyone will get the same construct)
Done in Embryonic Stem cells (HR in Embyronic stem cells)
How does IKMC KO genes - Construct used
- 5’ and 3’ homology arms (2 kb each)
- Neomycin antibiotic resistnce casstete (selection cassette)
- Needed because in Embryonic stem cells you need a selectable casete
- lacZ gene encoding B-gaactosidase
- Flp recombinase sites (Frt) that flank LacZ and neo
- LoxP sites flank the exons of the gene of interest (Have LoxP sites around exon 2 )
Use of LacZ in IKMC construct
Can use LacZ to understand endogenous expression
LacZ = encodes an enzyme that you can use as a reporter of gene expression (turn tissues blue when expressed)
How does IKMC construct cause a KO
To get KO –> target the 1st exon (when removed gives a frameshift) –> get null allele
- Need a frameshift so that when exon 1 and 3 recombine it is out of frame and there is nothing to code for
- When have a frameshift = code for nothing (get random nonsense)
- Because targeting 1st exon = don’t have to know anything about the gene –> just reomve the exon and get a null allele
WHY not remove the 2nd exon - 2nd exon might not give a frameshift = then removing the exon would not be interesting
LacZ in construct
LacZ has a splice acceptor in front of it (can accept splciing) AND LacZ as a stop codon
- NOTE - LacZ + splice accpetor is placed in an intron
Use of splice accpetor – INSTEAD of splciing norminal to fuse exon1 and exon 2 INSTEAD you splice from exon 1 to lacZ gene = make a null gene (null because of stop codon in LacZ)
Gene Trap
Gene trap = insterting things randomly into the gonome that will be controled by an already existing promoter and select for insertions that you want
Example - IN constrct lacZ is gene trap (Is inserted using a gene trap?)
First iteration of IKMC
First interation of the project where they tried to to KO all genes used a gene trapping stradegy –> BUT this is not used anymore
Why is this not used? - becuase it the gene trap created (with the splice accpetor before LacZ) COULD create a null allele (creates mRNA with ONLY exon 1 and lac Z spliced = null ; see in image) BUT it could also have alternative splciing
- Alternative splcing – subvert the null allele –> create a hypomorphrm (supress a small amount of gene BUT not all = might not make 100% null)
- ALSO a probelm with CRIPSR – when mutate gene in exon and make a framshft –> can create hypomorphs –> might not make a null (splice around the framshift mutations = don’t get null)
Limitation of IKMC construct
Limitation of the construct – since targeting all 20,000 genes and might no have prior knowledge of the gene –> the construct will always target exon 1 to create a frameshift for the rest of the gene
MEANS that if the gene has a lot of alternative splicing you can get a hypermorphic allele (here we are not interested in hypermorphic alleles we are intersted in KO)
NOTE - CHEKC IF HYPER or HYPO
Breeding mice outcomes
- Breed mice that express the construct
- Breed Mouse to Flip Recombinase THEN Cre recombinase –> get a conditional allele
- Breed to mice with Cre recombinatse –> creates a null allele with a reproter
Option – COULD make 1 reproter alele (with adding Cre first) and 1 floxed alles (adding Flp first)
Breed mice that express the construct
Breed mice that express the construct –> get homozygous mouse with the slice accpetor in front of lacZ –> get null alelle that has LacZ expression
- Homozygous mouse – both aleles expresses construct
BUT because of gene trap = could have alternative splciing = get a hypomoprh instead of a null
Splicing in Construct
Outcomes for splcining when have no recombinase added (mice is homozygous for the costruct)
Option 1- Use the Splice acceptor – splice exon1 and Lac Z (get Exon 1 – LacZ – Stop Codon – Exon 2 etc)
–> when translaed the proteins has protein with exon1 and lacZ
- Get the LacZ protein BUT not the rest of the gene
- LacZ here can act as a reporter
Option 2 - Don’t use splice acceptor –> splice exon1 and exon 2 = get normal gene expression (get protein)
- Bad becase this could resuce a gene we are trying to KO or could lead to hypomorphic allele (see some phenotype but less)
LacZ as a reproter
LacZ as a reproter – LacZ reprots on the gene (even when have KO) because have exon1 spliced to LacZ = get expression from the promoter of the gene = know the gene is being expressed BUT don’t actually get the exon2/3 translated because LacZ has a stop codon
USE – can see where LacZ is expressed (Shows where the gene that the consyuct is insetred in is expressed because the promoter of that gene is active to be able to express lacZ)
Breed Mouse to Flip Recombinase (Breed baseline mouse with teh orginal construct and breed to a mouse that expresses Flp)
Goal – remove the splice accpator and the neomycin cassete –> Go back to WT expression (get tranlsation of the protein because no LacZ with stop anymore)
When Breed to mouse that expresses Flp - LacZ and Neo get spliced out –> Result get exon1 – flp site - LoxP – exon 2 – Lox P (have 1 FRT and LoxP sites around Exon 2)
- IN THIS STATE = have WT expression and a floxxed exon 2
ONCE have this construct (with floxed ecxon 2) = can KO the gene
Use of Floxxed exon 2
CAN breed the mouse with a floxed exon 2 to a mouse that expressed Cre in a specific tissue or at a specifc time –> generates a null allele by reocmbining the LoxP sites
What exon is floxed after crossing mouse with Flp
Flox the first exon that when reomved would give a frameshift
Step AFTER you have floxxed exon 2
Because flox exon 2 –> breed the mice to Germline Cre mice –> remove exon 2 –> get framshift –> get Null alelle (creates a condition LOF)
Breed to Cre – Get Exon1 – Exon 3
- Once this is translated = exon1 and 3 translated and a premature stop codon because the roemval of exon 2 created a frameshift –> Creates a Null allale
Getting a true null with Flp/Cre
Would need to breed this to another mouse that has the same KO to make homozygous and get a true null
KO first allele vs. KO ready
PUT all together - Breed mouse to Flp then Cre
ONce add Flp – get exon1 – flp site - LoxP – exon 2 – Lox P –> Has WT expression with floxxed Lox P
Once add Cre - get exon 1 - Frt - LoxP - Exon 3 –> NULL
Breed to mice with Cre recombinatse (Breed mouse with origical construct with a mouse that expresses Cre)
Overall - creates a null allele with a LacZ reproter construct in the gene (KO reporter)
- Null because LacZ has a stop codon (not making the gene product BUT get lacZ being made)
- Can be useful to understand specific gene expression
When breed with JUST Cre - remove neomycn and exon 2 –> creates exon 1 – LacZ – exon 3
Step after Breed mouse with JUST Cre
After cross with mouse that expresses Cre - mice have 1 WT allele and 1 affected alelles –> Still have to breed to homozygosity so that both alleles encode lacZ
Once homozygous = have constutuve KO with 2 reproters
Putting is all together - Breed mouse with origical construct with a mouse that expresses Cre
Result of crossing mice with Cre or Flp
OVERALL – depening on the order at which you use Flp or Cre = can create different mice
- There are many things that you can do (useful + inexpensive)
Activity #1 - Design a mouse that expresses a reporter of the fuzzy gene (on Chr 1) when fluffu (on chr 9) is knockout in the skin
Goal - want to understand if the modulation of one gene affects the transcription of the other
- Trying to find the relatedness between genes (here seeing if fuzzy is effected when fluffy is KO)
Answer:
1. Breed Fuzzy allele mouse to germline Cre mouse –> Make the reproted (want it to be null)
2. Breed Fluffy Allelel mouse to germline Flp –> GIVES mouse that has a floxxed exon 2 (Has Wt expression of Furry with a floxed exon 2)
3. Breed Fluffy to WT and Breed Fuzzy to WT to backcorss and elimante the reocmbinases
4. Breed the Fluffy mouse and the Fuzzy mouse
5. Breed the mice to K-14 Cre –> Will reocmbine the LoxP sites flanking exon 2 in teh Fluffy gene = gets rid of furry (KO Fluffy)
Use of the KO Construct
Trying to find the relatedness between genes (here seeing if fuzzy is effected when fluffy is KO)
Example – if one of the genes is the transcrtion factor and the other is the target of the transcription factor –> THEN would want to know if have a decrease of increased of the target gene expressed based on that Transcription factor
Example of LacZ as reproter - using Fuzzy
Breed Fuzzy allele mouse to germline Cre mouse
Get null (not making the gene preduct BUT get lacZ being made)
Here LacZ can tell you if the fuzzy gene is being expressed (because promoter is active BUT won’t actually get fuzzy prtein vecause LacZ has stop codon so it is a null reproter
Screening for mutations - Scenrior – Core faciloty makes transjenuc mice by injecting zygotes –> they devlver 70 mice –> NOW What do you do?
NOW you need to screen to see which mice have the allele
Goal of screen – remove the WT mice (only want mice with desired edit)
Activity #2 – Design a genotyping strategy to confirm the mouse you designed is the mouse you have
Question – how do you design a screening strategy to find mice that have a floxed allele (image shows the target allele with LoxP sites around exon 3) - Background
Background – adding LoxP site with CRIPSR (CRIPR doesn’t have easy reporter or selectable marker and you don’t want to add in an extra reporter because wnat to add as minimal amount of things as possibel to mimic WT as much as possible)
- SsDNA that you add = has 100 Bp of Homology and the Lox sites (lox sites are 34 BP long)
Goal – Since we are trying to make a floxed allele we want to screen to see if Lox P has been inserted
Activity #2 – Design a genotyping strategy to confirm the mouse you designed is the mouse you have
Question – how do you design a screening strategy to find mice that have a floxed allele (image shows the target allele with LoxP sites around exon 3) - How will we screen
How will we screen –> Need to look for a 34 BP shift
Process - PCR amplify from an exon that we are targeting to the intron (intron is past the site of homology ; in image past the blakc line) (Can see green arrow primer pair and purple primer pair)
- Want to look for a shift in band size
Primers used:
1. for the 5’ lox site (shows if add the 5’ lo site)
2. for the 3’ lox site (shows if add the 3’ lox site)
3. Primer that bridges the whole thing –> shows deletion of the whole gene (deleted exon 3 that is floxed)
- NOT a different set of primer pair but uses the left most green primer and the right most purple primer)
Activity #2 – Design a genotyping strategy to confirm the mouse you designed is the mouse you have
Question – how do you design a screening strategy to find mice that have a floxed allele (image shows the target allele with LoxP sites around exon 3) - Results
KO lanes - Have smaller bands at the bottom (bands are from the prime outside of the exon – from the whole deletion primers)
- Shows in CRIPSR have effcincet KO
Flox lanes? - Some lanes have 2 bands – have a WT band and 34 BP increase in band size because inserted the lox site
AFTER gel = need to also sequence because there can be mutations in the Lox sites (can still get 34 BP but have mutations s lox site woudln’t work) or can lose some sequence in lox site
Efficiencey of CRIPSR
CRIPSR is more efficnet to KO using HDR than using HDR to insert something new = generally get a lot of KO
Process is so efficient that have mice that are homozygous for the KO
- IF had a lethal gene – all homozygous would be dead and would only recover heterozygotes
Goal #2 – Screen for a point mutation
Goal - screen for mice that have a point mutation (Screen for a difference in 1 BP)
How do you screen –> induce a restriction fragment length polymorphism
Restriction fragment length polymorphism
Overall - adding a Restricton enzyme site when you cause the point mutatiions (generating the point mutation of interest ALSO turns the site into a restriction enzyme site
Example – WT was TTCTACA–> with point mutations have TTCTAGA
- TTCTAGA is a Xbal Restriction enzyme cutting site
Process - Add Restriction enzyme sites into genes –> then PCR amplify gene –> Add Restriction enzymes
- PCR fragemnts that get cut are KnokIn (have the point mutation)
Issue with Restriction Fragment Length Polymorphism
Need to have a point mutations that would give a restriction enzyme site
If didn’t have a easy site – could use wobble sequence to change the DNA and add the restriction enzyme site BUT keep the same amino acid sequence
- Recode the wobble so can get restction enzyme site when introduce point mutations but keep the same amino acid
Overall - How do you screen
For a null allele – design primers such that you can see the deletion by PCR genotyping the offspring
For insertion – Can design primers where you can see insertion of a sequences (Ex. If insert GFP or GA tag or LoxP site)
What do LoxP sites add
LoxP sites add 34 bases to the normal intronic sequences
Issue with mice
Issue – there are a lot of biological processes where rodents are not particularly useful (mice are not humans – brains are different + different GI + humans have more genes)
Example – There are genes that humans have that mice don’t have + there are biological processes that mice dont have
Solution if mice don’t havea partciular gene or biologic process
Solution – If mice don’t have a gene or don’t complete a biological process THEN we can ise other non-rodent model organisms
Issue - Some biological processes that mice don’t have that humans have can be modeled in other species BUT those species might not have a lot of resources
Example Non-rodent system
Example – mequaces
Meqaces = closest model organism that we utilize
Use Meqaces for HIV reserach
- Mice are not useful for HIV reserach but Mequaces are a good model system
Issue with non-rodent systems
Issue with non-rodent systems – There are a lot of limitations of resources
Example – Generation time –> organism might make 1 offspring and it takes 5 years for that offspring to be sexually mature –> planning for the experiment needs to be long term
Means you might have specific biological limitations or reproductive limitations if you use non-geneticaly modified model systems
Human Pre-implantation embyro Vs. Mice Pre-implantation embyro
Normally have a pre-implantation zygote –> have sperm attached to the glycoprotein shell (glycoprotein shell = Zona pelusida)
Mice have 3 Zona palluseida protein BUT humans have 4 ZP proteins
ZP4 is not epxressed in mice but it is expressed in humans –> how do we study it –> ANSWER is use Rabits
- Could over express ZP4 in mice BUt doesn’t say what its natural function in
How do we understand how primate or human specific genes/ genetic elements are mediated
To understand primate or human specifc genes –> non rodent systems might be of more use
Example: Experiment – Using CRIPSR in Rabits to study ZP4
- Rabits have 4 ZP –> Use rabits to undersatnd what ZP4 protein does –> use CRIPSR to KO
CAN’T do this in mice because they do not have ZP4
Rabit experiment to study ZP4 approach + Results
Use rabits and KO ZP4 with CRIPSR
Results - When breed heterozygours with the ZP4 KO to get homozygous with ZP4 KO there are no pup
- Have pups in WT and in heterozygous BUT NO pups in homozygous KO (seen in bar chart) –> Shows ZP4 is important
Use of ZP4
Results showed no pups with homozygous KO BUT DO get fertilziation –> Means that ZP4 is not needed in fertilization becasue get fertiklzation with homozygous KO
Found that ZP4 is needed for structural integrity of Zona pelusida
- When poke the embyro –> KO has a floppy ZP vs. WT is more rigid –> shows ZP4 is important for structure of the zona pelusida
What do we needed to know to make a rabbit KO?
- Reproductive Stradegy
- Can you hyper-stimulate ovulation
- Can the species be synchronized (predictive syncronyzation)
- Genome annotation (refernce genome)
- Can you see the pronuclei
- Ploidy
- Embyronic development
Why do you need to know Reproductive stradegy for KO
Need to know Breeding efficiencey
Ex. Geroge church wants to bring back the wooly mamoth –> long gestation time
- Reproductive strategy precludes your ability to get germ plasm and reproductive function from an animal that gestates for 2 years
Why do you need to know if you can hyper-stimulate ovulation for KO
Can you hyper-stimulate ovulation – can you get enough oocytes in order to manipilate them
Example – for pigs – can pick up oocytes and fertlize in virto
Why do you need to know if the species be synchronized for KO
Example – we can make cows that have the same repdouction pattern so you know when to breed them/put embyoes in
- Can’t do with old world primate (don’t know how to syndrochrnize reproduction)
Why do you need to know Genome annotation (refernce genome) for KO
Need to know if the gene/the sequnece that you have is accesible
- Has become an issue with somatic cell engineering
Issue - If do a CRIPSR screen across the genome in cell culture against a human –> is the refernce genome and the genome in teh HelA cell the same –> ANSWER is maybe not they could have different sequences
- Means a percent of genes associated with CRIPSR screens don’t actually mutate the genome because the sequneece of the humans are different
Why do you need to know if you can see the pronuclei for KO
Need to be able to see the pronuclei because need to see he place where you will inject things (ex. Where inject CRIPSR)
Example – Can’t see pronuclei in pigs because oocytes are dense with lipids = you need to spin down so the pronuclei spin to the top
Where have KO been done
Have done KO in lizards + chickens + squals + pigs + non-human primates
Example – pig transplatatoon (pig organs into humans –> mutated 60 genes in order to do that)
- Use tranegsis and KO technology to solve organ transplant shortage
What happens sepcies that are difficult to get in large numbers
In species that are difficult to get in large numbers –> Efficiency of transgensis/mutation becomes key
Issue – CRIPSR is efficient BUT sometimes you don’t have enough germ plasm to make animals (I think to make transgenic animals)
Example #2 of using non-rodents
Example – Looking at human specific gene
Here we are asking what made humans (how did we gain brain volume) –> looking for new genes/genes that are expressed differentially
They found a new gene that is only in humans
Human specifc gene that was idetofied
They found a new gene that is only in humans
Gene was a duplication (Lost a splice site in the duplicated gene) –> in differential splciing enzymatic function was removed from the parent gene and removed the nuclear localization signal AND exposed a mitocondrial localization signal (1 nucleotide cnage in gene caused it to go rom nucleuas where had enzymatic acitvity to the mitocondria)
Example #2 of using non-rodents - Experiment to study human specifc gene
Expressed the gene in mice and in ferets (did selective expression) –> Showed stem cells in brains of mice and ferres expanded
THEN they did the same in a new world primate (Closer to humans but new world so removed)
- Needed this to be efficnet = Done using lentiviral mediate mutogensis –> THEN looked at F0 –> FOUND that F0 brains grew larger without needing to breed
Shows use of genetic systems to look at new genes and make a neomorph
Why did they not continue breeding when adding gene to new world primates
No breeding required because brains grew larger in F0 (cortex grew = terminated experiment)
Adding gene to new world primate
Only way to add the new gene to new world primate efficnctley is with lentiviral trangesis
IF Need this to be eeficnet = used lentivial mediated transgesis (lentrivial meidated transgsis = highly efficnet at transfering the genes = can look at F0)
Making Neomorphs
Adding a new gene = got a neomorph (adding activity from another species) –> neomoprh caused expanding cortext
