Lecture #7 - Mouse Genetics 1

Question 1

Ways that we can manipulate the mouse genome

Answer 1

There are a lot of ways that we can manipulate the genome of mice:
1. Random Mutogensis
2. Taregeted Approaches - Trangenic ise + KO mice + Conditional KO mice + Kockin Mice + Inducible expression
- Homologous Recombination = targeted transgenesis (put in specifc locus)

Question 2

Random Mutigensis

Answer 2

Overall - Characterize mutations ( just by watching mice that have mutated genes in colonies of mice
- Random mutations have been well characterized
- Can include ENU mutagensis

Example 1 – Mouse found at Jackson library because of its change in body weight
- We know a lot about the genetics of body weight because when we have a random mutation in mice during breeding you can see differences in body weight in colony of mice

IMAGE – comparing obese/obese mouse to KO if 1 signaling on brain –> see the animals phenocopy one another
- Can understand the mutation and where the effect of the gene is

Question 3

Example of Random Mutogensis

Answer 3

Mutations that have been mapped for coast color
- Easy to map coat color because you can see coat color

Shows natural mutation that are made and noticed in colonies of mice

Question 4

Issue in random mutagensis

Answer 4

Issue – it used to be hard to know where the mutation is in the germ line and how to map the mutation back to the gene

Issue until positionally cloning the gene that the gene

NOW we use sequencing technology

Question 5

ENU mutagensis

Answer 5

ENU mutigensis – take a male mouse and give a mutagen ENU and look for novel phenotypes
- Forward genetics

There have been large projects to mutate mice randomly using a mutagen
- Not done AS often in mice BUT has been done to find some interesting genes

Question 6

Applied genetics

Answer 6

Applied genetics - how do we manipulate the genome in order to understand gene function + gene regulation

Question 7

Transgenic Mouse

Answer 7

Over express genes

Manipulate the mouse genome by putting in exogenous genes
- Make transgenic mice by putting genes together and expressing in mouse

Usually done by injecting pronuclease

Question 8

KO mouse

Answer 8

Use of KO mice - important for understanding the requirements of genes in processes
- Remove genes from mice

When have a gene of interested –> the first things we ask if what is the KO phenotype because we want to know the requirement of that gene for any phenotype you are interested in

Now have multiple methods to look at gene function by LOF throughout the mouse genome

Question 9

Conditional KO Mouse

Answer 9

Conditional KO –> KO gene in specific tissue or under temporal conditions (only KO genes at certain times or place in mice)
- KO could be lethal or have non-cell autonomous effects

Ex. KO in the liver could lead to a nuerological defect
- Would not know for KO if this is autonomous or non-autonmous effect

Question 10

Knock in mouse

Answer 10

Can humanize mice gene by replacing a mouse gene with a human gene

Example – make a point mutations see what 1 nucleotode is doing

Question 11

Inducible Expression

Answer 11

Induce gene function to understand a graded effect of gene expression in some process we are interested in

Can make use of tet on/off inducible gene technologies to understand what the functions do

Question 12

Mouse development - Full Process

Answer 12

Overall - Fertikzies mebryoe goes from 1 cell –> 2 cells –> get compaction

Paternal and maternal genes come together to make a Fertlized zygote –> Pronuclear stage zygote –> 2 cells –> 4 cells–> 8 cells –> Blastomerre strats making E-cadherin and strats producing a morula–> start to get adherence junctions between the cells–> 16 cells stage (start to pump water into the center of the morula to generate a blastocele)–> blastocele
- Zygote = 1 cell embryo

Question 13

Compaction in mouse development

Answer 13

Compaction – indviual bastomere expresses cadherin and forms murula –> pumo in water In the center and make blastoeale (NOW have trophecatedemr vs. inner cell mass)

Question 14

Zygote

Answer 14

Zygote has a polar body has been extruded

Has 2 polar bodies – 1 poalr body has the entire diploid genome and 1 polar body has a haploid genome

Question 15

Pronuclear stage zygote

Answer 15

Pronuclear stage zygote – has paternal and maternal pronucleai –> both contain the entire haploid genome
- Patrenal is larger than the maternal (Sperm pronucleas deondenses and forms a haploid structure )

Use of pronuclei - Can inject DNA directly into the pronucleai to make transgenic mouse –> The DNA injected will encorprotae randomly into the genome (usually inserts into break)
- Injection of DNA can form cancatomeres

Question 16

Overall use of CRIPSR in mice

Answer 16

DNA injected in prouclei will insert randomly into the genome (DNA usually inserts into a break)

NOW have CRIPSR = make own breaks and put DNA here we want it to go = overcomes the randomness of DNA inserting into the genome

Question 17

Blastomere

Answer 17

Blastomaere = totipotent –> has the ability to make an entire organism

All of the individual balstomeres will produce an organsims

Question 18

Morula

Answer 18

Morula = compacted emryo

As morula progresses it starts to make adherence junctions between the cells

At morula stage we can’t seperate the cells anymore (they are no longer totipotent)

Question 19

Blastocele

Answer 19

Blastcoele = small cavity

Make the blastocele is the 1 event that creates the inner cell mass and traphectaderm
- Inner cell mass (ESC)= all the epithelial cells that will make the organism
- Trophectaderm will make the placenta and extra embryonic tissue

Want to manipulate the inner cell mass because inner cell mass contributes to the germ line
- Don’t want to manipulate cells in the trophectaderm because won’t get germ line transmission

Question 20

What cells in the blastoceoe are we interested in

Answer 20

Inner cell mass = the cells that we are intested in adding to/manipulating because they will make the next generation
- Inner mass = ESC –> make somatic tissue in mice

We can insert embryonic stem cells that we have manipulated outside of the mice and inject them into the blastocele cavity and SOME of those embryonic stem cells will generate tissue in the next generation and in the next germline

Question 21

Culturing ESC

Answer 21

Uses Inner Cell mass (Inner cell mass cells = Embyronic stem cells)

We can remove the inner cell mass cells (ESC) and can culture them

Culture the ESC – Take the epiblast and culture it onto fibroblast layer and manipulate the genes there
- Done in Vitro = can select for puromuycin resistant cells
- Can do transgenic or Homologous recombination

END - Once you have cells you can take them from culture and inject to blastocyste –> now have cells form a different mouse injected into a donor embryo –> makes chimera

Question 22

What can you do with ESC

Answer 22

Because we can culture the ESC in vitro we can manipulate hem

Example 1 – We can manipulate them by transfecting a gene in –> creates a transgenic mouse

Example 2 – Can replace a gene

Question 23

How do you manipulate ESCs

Answer 23

To manipulate ESCs = need to have a construct that allows us to select for specifc Homologous recombination

Example - Want to delete a gene in a specific location in the genome –> need long homolopgous regions on either side of the construct AND need to have a resustence gene

Goal - Want the construct to find the gene in the non-editted In the genome and use the homology arms to direct HR of the gene
- THIS SHOULD replace the orginal gene with whatever we are intesrted In

Example – Add a Neomycin reistnt gene

Question 24

Why do you need to add a neomycin resstnce gene

Answer 24

Need neomycin resistance because HR is a rare event = we want to select for this rare event

We can selevct for the rate event by selecting for neomycin reissnece

End - Have pure clones and then use the clone to inject into blastocytes –> NOW in the next generation we will have a deletion that we are interested in

Question 25

Second method of selection in mnipulating ESCs

Answer 25

Use a thymidine kinase selection motif --> cells become sensitive to democlovere Purpose - Selection of the construct with the selction motif incorporated Why would we add HSTVK – IF a construct integeates randomly into the genome then it with have the HSTVK gene and therefore everything that is integrated non-homologously/randomly throughout the genome will have this gene and we can select against that Have a minor effect of effincyeney of the process = not often used

Question 26

Homologous Recombination Method (process)

Answer 26

Process – take blastocyte --> culture them --> Mnipulate the blastocyte in culture by injecting ESC iinto blastocyte - Once select the population of ESC--> inject the ESC into blastocyte - To cause homologous recombination of a gene we use HDR --> constuct has long arms on each side of DNA – replace what is in the center of that with what we want - Usually have a marker that is addded when HR occurs that we can use to select for sucessfully manipulate blastocytes (important because HR is a rare event so it needs to be selected for) Eventually want to remove the seltable marker so so that it does not interfere with what we are intersted in

Question 27

Use of HR vs. CRIPSR

Answer 27

CRISPR = good inserting smaller things BUT if we want a larger manipulation then use Homologous recombination - HR = used for large constructs - CRIPSR = used for small mutations

Question 28

What does injecting ESC create

Answer 28

Inject ESC into pseudeo-preganant mouse --> hope you get chimeric mouse - Chimera = 4 parent mouse - Hope some of he cells with go to the germline (germline compatent) Skin of the mice show you if you have incorporation (Know you get chimeric mouse based on coat color) Done in129 backgroun (129 agouti color in 129 vs. balc 6 have a clack coat color) --> 129 allows you to use coat color to select for chimeras - Goal – want high chimeric (I think want a lot of Agouti) - Doesn't really care about coat color in F0 WHAT we really care about is if the mouse has the ESC DNA in the germline

Question 29

Chimera mouse

Answer 29

Chimera of cell will often be used from 2 mouse strains Example – cells are often from a mouse that has Agouti coat color are the cells are injected into a black 6 mouse (has a black coat color) ; ALSO Psudoepregant white mouse (foster mouse) In offspring the coat color can be splotchy (some of the coat color is black and some are Augouti form the ESC) GOAL- In the offspring you look for male mice that are highly chimeric (have a large proportion of coat color that is derived from the ESC injected cells) - Issue - Can have chimerism but no pups that have chimerism because all of the ESC incorporated to he coat NOT the germline (bad because you want to carry over to the next generation ) x

Question 30

Why do we want male chiemric mice?

Answer 30

1. Useful for breeding because they can breed with multiple females  2. Shows in the ESC incorporate into the germline 

Question 31

How do male chimeras show ESC incorporation in Germline

Answer 31

Male gives indictaion of chimerism in the germ line because if you inject male cells into the psudopregennat mouse THEN if the are signals in the germamline (menaing the injected DNA integrated into the germline) the you trun the mice male (XXY) IF male ESC incorporate into the blastocyte and intergarte into a germline vesicle (repdouctive tissue) THEN it will turn the chimeric mouse male because it will now have a Y chrosmome that is expressing SRY (turns the mice male) - If have enough SRY in the germ line = will turn the mouse from female to male = indicates that you have ESC that express SRY in the germline - If the donaor is XX and the ESC gave an XY then all of the offspring should be male if you get good chimerism within the germline

Question 32

What do you do with the chimeric mouse

Answer 32

Once have chimeric mice - breed the male mosaic mice to WT mice and get half of the genetics in the offspring derived from the KI (1 allele is WT and 1 allele is from manipulated ESC) WHOLE process takes a long time + is laberous

Question 33

Limitation of Blastocyte injection

Answer 33

1. Obtaining chimeras​ --> Mutation may not allow for mouse development 2. Chimeras going germline --> want to incorporate change to next generation - The injected ESC may not contribute to sperm development - Female chimeras are generally not good 3. Strain Choice  4. Maintenance of normal chromosome numbers - mES cell lines are prone to chromosome loss/gain --> in culture the chromsosmecan break or become annuploid (annuploid can't make germline chimeras) 5. Time and Money 

Question 34

Strain choice in Blastocyte injection 

Answer 34

The most common lab mouse (C57Bl6/J) is notorious for poor yield of chimeras and low % chimeras. For yeas they used 129 because they are germline compatent BUT the c57 were making chiemras but not germline compoenet Now have Black6 that is germline competent --> easier because need fewer backcrosses that you have to maintain

Question 35

Use of CRIPSR in Muce

Answer 35

Advent of CRISPR has made manipulating mice more straight forward and easier WHY – cutting the DNA with cas9 has make HR more efficient - Make a dsDNA break in a sequence specific manner - Can also have a homology vector --> can incprotae a ssOligoo and a repair vector (DNA break facilitates the incorporation of new seqeucne) NOW we can skip a lot of the selection with neomycin and can go directly to injecting components of CRIPSR into single cell zygotes

Question 36

Advangtage of CRIPSR

Answer 36

Decreases the time because there is no ESC No culture or chimerism or germline competancey because now you inject the pronuclear stage zygote with the gRNA and the tareting mechasnom and cas9 nuclease and repair vector

Question 37

CRSIPR process

Answer 37

1. Superovulation and breed--> get a lot of fertilized zyogotes at the pronuclear stage 2. Isolate single cells zygotes and microinject with cas9 compoenets --> Then culture to 2 cell stage embryo - Inject CRIPSR compoenents with gene of interest as gRNA into pronuclear zygotes - Inject cas9 + crRNA + tracerRNA + can add ssODN (ssOilogio) 3. Transfer 2 cell stage embryo to psudo-pregnant recipeinet and select - Reapir will happen and you can take the injected zygotes and transfer them Atto a pseuddopregnat female 4. Genotype pups to idetofy the desired alleles END – have 1/3 of ¼ of progeny that have the mutation

Question 38

At what point in CRISPr do you see what you want to se

Answer 38

Right from the F0 inject embyroes you can get most of what you are interested in (very effcicnnet) - F0 generation have mutation that you are intesrted ¼ of mice have the repair

Question 39

Use of CRIPSR

Answer 39

1. Single or double KO 2. Point mutations 3. Insertions/deletions 4. Floxed alelles 5. Knock in (exmaple GFP or HA epitope tag)

Question 40

Example 1 of what CRIPSR can make

Answer 40

CRISPR system is very efficient at making Insertions and deletions If we just have gRNA against the gene of interst --> Cas9 will cut DNA --> get INDELs during repair - Can get 1 or 2 alleles because the process is very effcicnet

Question 41

Example 2 of what CRIPSR can make

Answer 41

Can do small repairs Example – Do a knock in where you have a single point mutations within a gene - Can add a 100 nucleotide ssOligo (co-inject the olgio with cas9) --> CRISPR will repair the cut with the ssOligo in homologous manner such that you replace the orginal DNA with whatever nucleotide you want changed

Question 42

Example 3 of what CRIPSR can make

Answer 42

Can add a larger oligio --> can cut on either side of the exon and then repair with a larger construct Example – Can put Lox sides on either side of the exon to make a conditional allele

Question 43

Limitation of CRIPSR

Answer 43

Every time we have a longer piece of DNA that we have to repair = it becomes less eficincet (need to screen more mice) BUT it is still more efficient than HR There is a size limit and complexity limit that we can use CRIPSR - Because of this there is still use for HR using ESC (use HR for larger constructs or things with repeataive sequences that we can’t make ssOligios for)

Question 44

Cre recombinase (overall)

Answer 44

Overall – Use recombinase to mutate gene and see what the autonomous function of gene is in tissue/cell Cre recombinase = uses Lox P that you place on either side - Have a exon or gene between 2 loxP sites - LoxP is reconized by Cre - When recombination occurs it forms a circle that the cell will exclude

Question 45

Expression of Cre

Answer 45

Can express cre in Trans Can encode Cre in RNA in virus or can encode in genome - As long as you get the protein expressed in cells of interst THEN you will recombine the LoxP sites

Question 46

Example use of Cre

Answer 46

Example Use – Might not want to KO a gene because it is an essential gene or it is used for embyrogensis --> KO these genes is not be useful if you are trying to figure ut what gene is doing in an adult tissue

Question 47

LoxP site facing in the same direction

Answer 47

If the lox are facing the same direction then they will delete the gene when they see Cre recombinase - The 2 loxP sites get recombinied --> lose of whatever genetic element is placed between the 2 lox sites - Works over long distances Useful beceuase there are a lot of reagnets in mice where we can express Cre in different tissues or under different timings = we can KO a gene in specific time and in a specific tissue

Question 48

LoxP site facing in the opposite direction

Answer 48

If teh LoxP sites are facing towards each other --> flip the thing between the Lox sites so they now go in the opposite direction - Invert the middle sequence - Works over long distances = can flip very large segments of the chromosome (example making balancer chromsomes where half the chromosome is flipped) Purose - Useful if you want to activate a gene under certain circumstances OR if we want a null at some point and then want to flip back to WT in adult

Question 49

Limitation and Use of Cre

Answer 49

Not limited to 1 seqenece – can recombine across chromosome - Can flip or KO a giant peice of chromosome There are different sequenves that are recognized by Cre but won’t recombine with each other - Example – Lox 66 is recognized by Cre (can use it like LoxP) but it won’t recombine with LoxP --> now have multiple recombinse events that are all recognized by Cre but used in different ways Use = making stochastic reporter

Question 50

Seqeunces recognized by Cre

Answer 50

Have Class LoxP sequence BUT there are different kinds of heterotryoic sequences IF make small mutations in the lox sites = can get different lox sites that are also seen by Cre BUT do not recombine with each other Example – LoxP and Lox71 don’t reocomcbine BUT LoxP and LoxP recombine --> SO you can have multiple different lox sites within 1 construct within 1 gene and they won’t recombine with the hetrotypic site BUT they will recombine with each other - Using the different sequences of lox sites allows you to do complex things

Question 51

Heterotypic Lox Sites

Answer 51

Use - Stoachstic reporter Example – marks neurons and follow neurons in development - Designed a vector – transgenes express RFP or YFP or Cyan - using interventing lox sites --> Upop Cre activity you recombine in stochastic manner (random) to express RFP or YFP or Cyan - Different lox sites don’t recombine with different sequence but can with the same sequence - Example – Have Cre and get LoxN – get RFP and delete everything else beceuase removed lox22 and lox72 and loxp sequnece Shows how use different lox sequences to effect gene expression

Question 52

Answer 52

Two sequences that face towards each other but then when flip the you can NOW delete the Lox site Doesn’t matter which lox site Cre chooses first

Question 53

AAV

Answer 53

Flex vector = AAV Can sue a flex vector (AAV) for Cre AAV = uses the same technologies to turn gene on/off in a tissue specific manner - Express Cre and add the construct (image) to express genes with AAV - Use AAV because have size constraints – don’t need complex genetic control just flip on/off = more compact)

Question 54

Pre-flipping exon

Answer 54

Use - Can see when the effect takes place – informs how you attack and therapy and when the pertivasion is required Example – Gene that causes Rhett syndrome – can you reverse this? Question - Is it a permanent developmental change or is it something you can effect in the adult (is there therapy that can be direct at the gene to make kids better) - To answer - Did they by making the exon backwards = have a null allele (same as deletion of exon) = can flip on at any time in adult - Showed this is not just a developmental disease BUT makes the mice normal BUT turn the gene back on in adult - NOW not just developmental affect where you targeting developing humans but can also affect expression in adult where you diagnose them and can have a therapy for them - Can diagnose and have targeted theapry

Question 55

Use of CRe and Pre-flipping and exon

Answer 55

Using this when have developmental effects Can see when the effect takes place (if before birth have therapy BUT if in embyro then can’t make thearoy) --> informs how you attack and theapy and when the pertivasion is required

Question 56

Issue in gene therapy

Answer 56

We know single gene mutations for many disease that we know how to reverse in people easily BUT we don’t know how to do it safely in humans Issue = NOT the effect of gene but in the off target effects - Reason why don’t work with F0 population when target these genes -> because we want to backcross so we have WT everything else and only effect the thing we are interested in - Targeting DNA is always an issue (always a toxcity involved)

Question 57

Issue with gene therapy + liver

Answer 57

Issue in gene theapry = always get hepatoceullar damage - Can deliver genes to organs BUT it never works in humans because of toxicity Hard when have human specific toxicity because there is no way to model it

Question 58

Cre mediated Inversion 

Answer 58

Using inversion to flip genes around Can use lox site to flip entire chromsomes (Ex. Balancer chromosome in flies) Can add extra chrosmome in mice - Used to study trisomy --> make model where duplicate entire chromsomes to understand dosage of chromosomes

Question 59

Resources for Cre recombinase use

Answer 59

There are many resources for Cre recombinase use Example – Jackson lab has Cre expressing mice (Ex. have liver expression or brain expression) Example 2 – have Genstat program that make BAC transgenic mice Can also make Transgenic with specifc promoter

Question 60

BAC expression

Answer 60

BAC = often used in transgenics (Ex. repositories in nueroscience) BAC transgenic mice = use a bacterial artificial chromosome so that the regulatorty element for the gene is very large - regulatory elements is larger than in transgenic – transgenic uses a minmal promoter and mimal enhancer to direct gene expression Example – want expression in a very specific population of neruons = often requires more regulatory elememts

Question 61

Genestat

Answer 61

Genestat = generate tons of mice that express EGF and Cre in many tissues in the nervous system - Gensat = made BAC that express Cre and GFP Use of BAC that express Cre and GFP = do electophisiology of specifc population or manipulate population

Question 62

Knock in Cre

Answer 62

Can Knock in Cre within a gene/locus = get the exact expression of Cre in the gene that you are interested in - Used if you have a specific gene expression pattern that you want to manipulate genes in

Question 63

How would you express Cre from endogenous allele BUT not affect the allele

Answer 63

How do you make 2 genes out of 1? Answer: 1. IRES – get 2 proteins from the same locus 2. Knockin to a locus that has normal expression and can still express Cre

Question 64

Temperal Experssion of Cre

Answer 64

Use tetracyline or Tomaxaphin BOTH have caveates - Ex. Tet inhibits mitochondrial function and Tomaxaphin has off target effects in estrogen signlaing + Tomaxaphin is toxic to Adipocytes

Question 65

CreERT2 Tamoxifen

Answer 65

CreERT2 Tamoxifen regulates CRE Ert --> leads to a tomaxin inducible Cre reocmbinase - Have estriogen repctor that has been mutated so that it only interacts with Tomaxaphin + Cre has been used to ligand binding domain of estrogen receptor Overall - Expression of Cre is inative in the absence of Ligand --> Only get recombinase in the presence of tomaxafine Because ER does not bind to estrogen + Cre is bound to ER --> THIS seqeuesters the fusion protein and therefore recombinase in the cytoplasm (while the estrogen rector is attached to Heat shock proteins)--> when the tomaxphin binds to the receptor the Heat shock proteins dissociate and the receptor can go to the nucleas and access DNA and catylyzes recombination with LoxP

Question 66

Use of tomaxaphin

Answer 66

Useful for genes were you want the mouse to develop fully - Do in an adult mouse and have some kind of manipulation before you remove the gene Example – make the mouse diabetic or obese and then understand the therapeutic role of removing or activating a gene under certain conditions Can be done with any steroid receptor (Example Gluccoicoid or progesterone receptor)

Question 67

tetrycyoin responsive Cre

Answer 67

Two part system to express recombinase: 1. tet responsive promoter --> Promoter with multimerized TRE upstream of transgene 2. Tissue specific rtTA (making TF) Overall - Have tet responsive elements that binds to Tet activator (rtTA) and expresses Cre in the presence of Tet - rtTA = binds to tet repsonsive element End - get expression of Cre in a tissue specific manner - Tissue specific because you have to expression of tet responsive protein (rtTA activator) from a tissue specific promoter - Instead of having tissue specifc promoter expressing CRE NOW you expess rtTA via a tissue specifc promoter and rtTA ONLy becomes activated when have tet

Question 68

rtTA

Answer 68

rtTA = Transcription factor --> binds to the tet resonsinve element on the promoter - rtTA = only binds to TRE if have Tet Tet = gene ON

Question 69

Response of Tet system

Answer 69

Responds to how much ligand you put in In Tet = can express things in a dose dependent maner (add more Tet = get more expression) Compared to tomaxaphin - Hard On/Off based on if recombinase has access to nuclease

Question 70

Issue in tet system

Answer 70

Can get leaky expression (get some expression of transgene = bad - Know there is a lot off target effects Experiment: - TF binds --> Express the TRE --> get expression of the downstream Cre - When have no TF – bred mice to mice that express Toxin --> See if mice die or not - See body weights decrease + see half the mice die = have off target effects - Hard to see off targets normally – need to put in a reporter allele + need to be thorough in looking at reproter BUT here it easy to see of targets because see if alive or dead See there is enough endogenous expression of Cre to affect experiment that you might be doing - Get off target effects in the absence of tissue specificity

Question 71

Transgenics

Answer 71

1. Direct Injection of Linear DNA 2. BAC transgenics 3. Targeted Integration 4. Viral/retrotransposon expression

Question 72

Direct Injection of Linear DNA

Answer 72

Directley inject linear DNA into an embryo or into a zygote (inject in pronucleus) Mediates random insertion --> likely will go somewhere with a break and fills the break in Forms concatermers (NOT good if you require only 1 copy of your transgene )

Question 73

Issue with random insertion

Answer 73

Issue with random insertion of transgene = lands in random locus --> means you need to screen many lines/need many lines to test in experiments beause have many different inserions Example – can land in the middle of the immunoglobulin gene

Question 74

BAC transgenics

Answer 74

BAC = large construct that carrys a lot of enhancer elements in order to specifically express your transgene of interst - Large regulatory elements Use - If you need more regulation upon expression Random insertion BUT usually only 1 insertion because it has a large peice of DNA

Question 75

Targeted Integration

Answer 75

Put DNA specifically within a locus by HR or CRIPSR Can put use targeted integration and put gene in safe harbour locus

Question 76

Safe Harbour Loci

Answer 76

Safe harbour Loci = not affected by other regulatpry elememts and are constitutive Use - Place a gene in a safe harbour locus and know there will not be large effects on other genes - Under random integration = we would worry about effects on other genes - Can use to put in transgene that has more stable expression = put in different safe harbour locus Example - Place gene in ROSA26 using CRIPSR or HR to express it - R26 = highly expressed BUT there is not phenotype when lost

Question 77

Issue in Random integration

Answer 77

A lot of transgenes that have been randomly integrated within the gene can disrupt genes which can affect phenotypes indepentley of what you are interested in

Question 78

ROSA26 Locus

Answer 78

Purpose – used if you want to express transgene - If want to add 10X a gene in a tissue specifc manner--> put in ROSA locis so that you don’t affect other genes and express the gene in a straight foward manner The gene of interest is targeted into the first intron of the locus The expression of the gene can be driven by endogenous ROSA26 promoter or by a stronger promoter OR Expression can also be stimulated via Cre recombination – removing the selection cassette.​

Question 79

Viral/retrotransposon expression

Answer 79

Lentivirus Single copy --> Places 1 copy into the genome randomly (Pseudorandly for lentiviral) Good if you want a single copy and high effcicnecey transgenesis Many ways to express proteins

Question 80

Answer 80

Skin specific promoter upstream of Cre – Fuzy has two loz P sites with Stop codn between --> controlled by a consttutive promter THEN at 9 weeks add tomaxphin = flox out stop codon and get fuzzy/fluffy,eGFP expression - Put n Y chrosome (don’t really need to you could just pick out males) - Skin specifc promoter driving Cre – add tomaxaphin and turn on transgene

Question 81

More than just Cre

Answer 81

Question 82

Mouse repositories

Answer 82

Have many repositories where you find mice that have been generated Example 1 - have conseersium that is trying to make floxed alleles for al genes Example - Jackson Lab has mice

Question 83

Nomenclature in Mice

Answer 83

B6 – mouse strain that you are currently in (backross on Blass 6) 129 – Strain made on (orginal mouse backgroun) Gt(ROSA)26 - Gene trap in Rosa 26 locus Cre – Trasngene tm1 - trageted mutations J – J form (substrain) Tyj – Taylor Jacks made it (creator)