19.+20.+21. Mice genetics

Question 1

What is forward and reverse genetic analysis?

Answer 1

Forward genetics - phenotype driven:
random mutation -> phenotype -> gene identification -> interpretation of gene function => uncovers genetic basis of phenotype

Reverse genetics - gene driven:
gene identification -> targeted mutation -> phenotype -> interpretation of gene function => uncovers gene function by targeted mutations

Question 2

What is gene trapping?

Answer 2

Gene trapping - an approach to study gene function using random insertional mutagenesis introducing a tag to identify (an intermediate between forward and reverse)
Random tag insertion -> gene identification -> phenotype -> interpretation of gene function

Question 3

What are the three approaches for studying gene function in vivo?

Answer 3

• Forward genetics: random mutation -> phenotype
• Reverse genetics: trageted mutation -> phenotype
• Gene trapping: random tag insertion -> following gene in phenotype

Question 4

What is an example of a conserved gene in species development?

Answer 4

Pax6 for eye development with conserved am. a. across species

Question 5

Explain what is a balancer chromosome

Answer 5

Balancer chromosome: engineered chromosome which prevents recombination during meiosis ensuring specific genotypes - because genes are inverted

Question 6

How are recessive mutations studied?

Answer 6

Ex: Heidelbrg screen in Drosophila - forward genetics: crossing a fly with balancer chromosome + random mutation -> crossing until mutated recessive allele is segregated homozygous genotype -> can study recessive phenotypes

Question 7

Explain the Heidelberg discovery

Answer 7

Heidelberg discovery - Hedgehog (Hh) pathway: Hh mutations in flies cause forebrain defects: decreased sonic hedgehog -> no separation of forebrain

Question 8

What are the possible chemical mutagens?

Answer 8

• ENU (ethylnitrosourea)
• EMS (ethyl methane sulphonate)

Question 9

How are zebrafish large scale screens performed?

Answer 9

Random mutation -> crossing until affected genotype observed as phenotype

Question 10

Why are mice a suitable model for studying mammalian developmental processes?

Answer 10

Mice are suitable model species because:
- mammalian
- small size
- diet + environment
- reproductive efficiency (non-seasonal breeding) - av litter size 10, gestation 20 days
- long term record + DNA resources + sequenced genome
- tolerant to inbreeding
- embryos and sperm can be cryopreserved
- ES cells
- publically accepted for experiments

Question 11

What are the apparent differences between mouse and human genomes?

Answer 11

Mouse genome: 19 autosomes, telocentric chromosomes, 37k CpG islands

Human genome: 22 autosomes, metacentric and submetacentris chromosomes, 45k CpG islands

=> 70-90% sequence homology - suitable to be used as models for human processes - syntenic genes (almost complete synteny on X chromosome)

Question 12

Explain the way in which ENU functions

Answer 12

ENU - supermutagen:
- transfers ethyl group to oxygen + nitrogen radicals in DNA -> mispairing
- ex: single base pair substitutions in spermatogonial cells at high efficiency
- each F1 generation may carry up to 100 mutations (if one locus mutated - can impact many phenotypes)

Question 13

Missense vs nonsense mutation

Answer 13

Missense mutation: am. a. substitution

Nonsense mutation: premature protein termination - stop codom

Question 14

Hypomorph vs antimoprh vs neomorph alelles

Answer 14

Hypomorph: mutant allele retains some gene function, less severe than LOF

Antimorph: mutant allele that antagonises normal gene function

Neomorph: mutant allele acquired new function

Question 15

What are the genotypes of inbred and outbred mouse strains? What are their features?

Answer 15

Outbred:
- approximates human population
- heterozygous vigour
- maintain mutants as heterozygous - don’t die

Inbred:
- unique strain
- brother x sister >20 generations
- fixed genetic background - high probability of homozygosity - every individual is identical for autosome genotypes (X and Y diff)
-> mutations can be studied because background is fixed - other genes won’t interfere

Question 16

Explain what is genetic linkage?

Answer 16

Genetic linkage: association of alleles which influences them to get transmitted to offspring in parental combination more frequently

Question 17

How is recombination frequency influenced by distance between genes?

Answer 17

The further apart - the higher rate of recombination on the chromosome

Question 18

Explain what is serial backcrossing?

Answer 18

Serial backcrossing - allows to determine which gene is responsible for a specific phenotype:
cross with inbred strain -> introduced new trait into neutral background -> cross offspring with parent again -> congenic strain created

Question 19

What are the two commonly used DNA components used as markers?

Answer 19

Microsatellite markers - dinucleotide repeats (ex (CA)n where n=10-60) distributed in the genome - repeats are varying between mouse strains

SNPs - specific shared between strains but also can vary - can be used for identification

Question 20

How can DNA sequences be identified that stay with the trait after multiple rounds of backcrossing?

Answer 20

Identifying DNA sequences that stayed with the allele for specific trait:
1. Design primers for PCR
2. Amplify by PCR
3. Sequence PCR products
4. Compare sequences from control vs trait affected

Question 21

How can functional gene identification be performed?

Answer 21

Functional identification:
- backcrossing - gene region of the trait identify with markers (microsatellites / SNPs)
- gene region cut into diff segments - diff segments inserted into diff BACs
- diff BACs transformed into diff mice
- identify which BAC produced the target phenotype - BAC complementation

Question 22

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Question 25

What are the stages of early development?

Answer 25

Fertilization -> morula -> blastocyst -> embryo

Question 26

What is are the processes that lead fertilised egg development into implantation stage embryo?

Answer 26

- Ovulation - Fertilization - Early cleavage - Compaction - Hatching - Implantation

Question 27

Explain what is pronuclear transgenesis?

Answer 27

Pronuclear transgenesis: - Collect **fertilized eggs** from mother I - Inject **male nucleolus** of fertilized egg **with** purified **target sequence** from BAC/YAC (fragment amplification) - **DNA inserts randomly** - **Transfer into** mother II - **pseudopregnant** (mated with sterile males) - Observe **phenotype** in born **pups**

Question 28

What tool is used for pronuclear transgenesis?

Answer 28

**Inverted microscope** with 400x magnification with **micromanipulator**: **right** - **injection pipette**, **left** - negative pressure **holding pipette**

Question 29

What are the potential effects of new gene introduction via pronuclear transgenesis?

Answer 29

- **Position effect**: **integrates randomly** - **chromatin state** determines if will get **expressed** / **not expressed** - **Mutagenic**: integration may **disrupt** a **gene** - mutation - **Concatemers**: injected DNA can **recombine** and **form concatemers** before integration (usually introduced as multicopy arrays) - **Not intact**: injected DNA may be **partially degraded** - not all sequence integrated - transgene **insertion not intact**

Question 30

What are concatemers?

Answer 30

Concatemers: a **continuous DNA** molecule that contains **multiple copies** of the **same DNA sequence** linked in series

Question 31

Why is each strain of pronuclear transgenesis unique?

Answer 31

Each strain of pronuclear transgenesis is unique: - i**random integration** - diff integration sites - **different copy numbers** (may be degraded/ recombined - change lengths) - **integrity** of transgene may **vary** because not all will be functional - can insert into **heterochromatin** / be **mutagenic**

Question 32

What are the two most commons reporters in developmental biology?

Answer 32

LacZ and GFP

Question 33

How can chimeric organisms be produced?

Answer 33

**Plasticity** in early embryos - can **combined** **cells** from **2 diff embryos** - form **chimera** - develop into chimeric animal

Question 34

Explain the structure of the blastocyst

Answer 34

Blastocyst: - **epiblast** -> fetus - **hypoblast** -> yolk sac - **trophoblast** -> placenta **ICM** = epiblast + hypoblast

Question 35

Which structure are ES cells derived from?

Answer 35

**ES cells** taken from **epiblast** (develops into fetus) - **pluripotent** - capacity to differentiate into **all germ layers**: ectoderm, endoderm, mesoderm However, can ES cells can give rise to tumours - **teratocarcinomas** if **injected** into **random locations** (ex under skin)

Question 36

The schematic of embryo development

Answer 36

Question 37

How are ES cells derived and cultured in vitro?

Answer 37

ES derivation and culturing: mice mated -> **blastocysts** taken out -> blastocysts **cultured** on **feeder cells** in vitro -> subculture by **separating** -> necessary **signalling molecules** to **remain** **pluripotent** / **induce** **differentiation**

Question 38

How are chimeric animals made with ES cells?

Answer 38

1. **Blastocysts** isolated from mice strain 1 -> **ES cells** from strain 2 **injected** into blastocysts 2. **Implant chimeric blastocysts** into **pseudopregnant** mice -> birth **chimeric** **pups** => but **not all will be chimeric** - not all transgenic because - **not all ES will integrate**

Question 39

What are endogenous genes?

Answer 39

Endogenous gene: gene **originating** **from within a living system** - in contrast - **exogenous** - originate **from** **outside** (transgenic)

Question 40

How are founder transgenic mice strains obtained?

Answer 40

**Chimeric** pups from foreign ES transgenesis are **bread** **with** the **original strain** type (the strain that provided the blastocysts - genetic background) -> bread **until chimeric genotype isolated**

Question 41

How are ES cells genetically manipulated?

Answer 41

1. **DNA vector designed** for **HR** with the **target gene** 2. The vector can be **integrated into ES** cells by: - **Gene targeting**: via **HR** - only integrates if **homolog sequence** found - **HSV-tk not integrated** - **Random integration**: **insertion** into **random location** - **HSV-tk integrated** - will be **screened** and eliminated in the experiment - select against random integration

Question 42

What are the two types of genetic vector integration into mouse genome?

Answer 42

Vector integration can be: - **Gene targeted** integration - **Random** integration

Question 43

What are the parameters important for gene targeting in vector integration?

Answer 43

**Parameters** of **vector** for **successful** targeted **integration**: - **length of homology** - the longer the higher freq of integration - size of non-homologous sequence doesn't make difference - **Isogenic DNA** constructs **increase integration freq** -> using non-isogenic DNA causes mismatches

Question 44

Explain what is isogenic and non-isogenic DNA

Answer 44

**Isogenic** DNA: **sequences** that come from genetically identical / nearly identical organisms (ex from inbreeding) - **very similar** / **identical sequences** **Non-isogenic** DNA: **sequences** that come **from genetically different organisms** within species - between species

Question 45

Explain the experimental approach of gene targeting

Answer 45

Gene targeting - **specific integration** of vector into **ES cells** 1. Introduce **vector** into cells - adding **selection markers** - **screen for cells** based on introduced **selector** (ex ganciclovir) 2. **Replate** resistant colonies - further **culturing** in well plates 3. **Identify** inserted **gene** by **Southern** blot/ **restriction enzymes** + **Southern** blot 4. **Freeze** other cells for later / use for chimera production

Question 46

What experimental technique is used to screen for targeted clones?

Answer 46

**Southern** blot: **probes** for specific target sequence **hybridisation** - s**ee if integrated** into cell genome **Restriction enzymes**: will **identify change** in cut endogenous **fragment size** if integration occurred - **confirmation** that the **specific target** sequence integrated - by **Southern** blot hybridisation **PCR**: **primer designed** within transgenic **target sequence** + **primer** for the **sequence** from **other side** -> **if** targeting **successful**, **PCR will amplify** the sequence using the primers and produce **specific length products** **if** **random** integration - the **products** will be **longer** than from gene targeting

Question 47

How can a specific gene expression pattern be investigated?

Answer 47

Inserting **gene reporters** into the target gene locus - **LacZ** / **GFP** - **fused in frame** to the **upstream** locus - **integrated** ino genome **via HR** => instead of exons 3, 4, 5, 6 - **some parts deleted** - the target vector inserted instead in recombination - could lead to **problems if** they are **regulators** / **ORF** -> target vectors can also be integrated without deletions

Question 48

What is a knock-in (KI) vector?

Answer 48

Knock-in (KI) vector: a **cDNA** fragment **integrated** into genome **to be expressed under control** of an **endogenouse gene** - ex reporters (LacZ, GFP) - controlled by promoter of an existing developmental gene

Question 49

What are multicistronic constructs?

Answer 49

Multicistronic constructs: **genetic constructs/vectors** that carry **multiple genes** within a **single transcript** - coding **ORFs are linked** and **transcribed together** as **single mRNA** - **simultaneous expression** of multiple proteins from one transcript Consists of: **shared regulatory elements**, **IRES**, **2A peptide** sequences

Question 50

What is the structure of multicistronic constructs and what are the roles of each component?

Answer 50

- **Shared regulatory** elements: simultaneously controlled expression - **IRES**: allows **CAP independent translation** mRNA into proteins - **2A peptide** sequences: regulates **expression** to be **at equal levels** of each **cDNA**

Question 51

Explain homozygous gene targeting

Answer 51

Homozygous gene targeting - 2 ways: - **using same vector** as gene targeting but using **different selection markers** for same allele - **using resistant casette** - **increased levels** of the **first selection marker** gives **both target alleles** ??? dont get

Question 52

How are heterozygous / homozygous gene targeting products selected?

Answer 52

Most integrations random - **gene targeted integrations** in ES can be **selected** using **drug selection** - which don't die replated - purified colony obtained - targeted genetic **integration checked** in DNA analysis (**Southern**) - **ES lines** used **for pronuclear transgenesis** - **backcross** to **obtain transgenic strains** -> **intercross** to **obtain** **homozygotes** / **heterozygotes**

Question 53

How is gene targeting used for gene function analysis?

Answer 53

1. **Reporter** incorporated into target gene 2. **Pronuclear transgenesis** 3. **Observe reporter** in offspring - **lineage tracing** + localization

Question 54

What are KO phenotypes?

Answer 54

**Knock-out** phenotypes - **gene function disrupted/altered** - **effects** **observed** in offspring using **reporters** for that gene + others (to visualize structures that will be affected)

Question 55

What is gene redundancy?

Answer 55

Gene redundancy: **in gene KO** **other genes** can take over and **compensate** for the knock-out **function**

Question 56

How can experiments be modified if gene knock-outs don't work because of gene redundancy?

Answer 56

In **gene redudancy** loss of gene function is **compensated** - instead of knock-out **edit for** **gene overexpression** - observe effects

Question 57

What are the transgenesis applications in mice?

Answer 57

Question 58

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Question 61

What is the modern approach to transgenesis that ensures gene targeting?

Answer 61

Generation of ES cells by HR - a lot of work + time consuming -> **CRISPR-Cas9** used - Cas9 **endonuclease** - **guide RNA** (gRNA) - **target sequence** - protospacer adjacent motif (**PAM** "NGG")

Question 62

How does CRISPR-Cas9 cut and what are the components?

Answer 62

Components: - **Cas9 endonuclease** - guide RNA (**gRNA**) - contains **20nt protospacer** (target seq) - designed **upstream of** protospacer adjacent motif (**PAM**) - **Cas9 recognises** PAM - **cuts** both strands **3nt upstream of PAM** Protospacer adjacent motif (PAM "NGG")

Question 63

How is a sequence integrated into genome after CRISPR-Cas9 cut?

Answer 63

CRISPR-Cas9 **cuts both strands** 3nt upstream of PAM - cut **repaired** by: - non-homologous end joining (**NHEJ**) - for gene knock-outs - **unpredictable result** - homology directed recombination (**HDR**) - **introduce specific mutations**, reporter genes - **precise** insertion/modification - cell repairs using **insert as template** - **two gRNAs** target sites chosen - will **delete** a **larger** genomic **region**

Question 64

How can CRIPR-Cas9 be used for pronuclear transgenesis?

Answer 64

Cas9 and gRNA **injected into male pronucleus** - repair via **HDR** **integrating target sequence** - **blastocysts** **transferred** into **pseudopregnant** mother II However, **off target effects possible** - Cas9/guideRNA **cleavage not always precise** - need to **choose target sequences** with **minimal homology** to other genes / regulatory sequences

Question 65

What can be used to reduce off-targets effects of Cas9 and guideRNAs?

Answer 65

- **Choose target sequences** with **minimal homology** to other genes / regulatory sequences - **Mutant Cas9 nuclease** (Cas9D10A) - **cleaves** only **one strand** -> using t**wo gRNAs specific for each strand** in the double strand cleavage + **Cas9D10A** generates an **overhang** -> reduces off-target integration Unlikely that there is another site with 2 specific sequences that create exact overhangs - reducing the chance of ds breaks leading to incorrect integration

Question 66

What is conditional gene knockout?

Answer 66

Conditional gene knockout - technique that introduces **KO in a specific** **organ**/ **tissue**/ **cell** at a **specific time** To **investigate** diff **gene expression** properties **controling**: - **time** of KO (ex for KO not to be lethal / to investigate gene role in developmental stage) - specific cell (**localization**) KO - **permanent tag** (reporter gene - track lineage) - **mutation** (activate mutationn at given time/tissue)

Question 67

How to induce a deletion in conditional genetics experiment?

Answer 67

**Site-specific recombination** (SSR) In-vivo - **inducing mutation** (deletion): - **insert loxP** sites **by gene targeting** to **flank target sequence** - **insert Cre-recombinase** expressing transgene -> **recombines** the **sequence flanked by loxP** -> DNA **excised**

Question 68

How to induce a deletion at a specific time in conditional genetics experiment?

Answer 68

If deletion required at specific stage in development or adult -> use **inducable deletor** ex: **Cre-ERT2** and **tamoxifen**: Cre-receptor construct Cre-ERT2: **Cre fused to mutant estrogen receptor ERT2** - **responds** to synthetic **analogue** of **estrogen** - **tamoxifen** -> upon addition of tamoxifen **Cre moves** to the nucleus - causes **recombintation** -> gene **excision** (deletion) Time when tamoxifen added - chosen freely

Question 69

Exlpain the experiment how was lineage tracing of Lgr5 cells in intestine lining crypts investigated?

Answer 69

2 transgenes - **LacZ** and **CreERT2/tamoxifen** - allowed to **compare expression** in **different time points** of development - expression of LacZ induced by tamoxifen addition time point **Lgr5** **expressing** cells - **stem cells** - the stem cells also inserted with **LacZ** - when **differentiated** - no longer stem cells - can be **traced by turning on LacZ** reporter - **Tamoxifen** addded **day 1** - **Tamoxifen** added **6 months** -> **previously Lgr5 expressing** cells **occupied** the whole **crypt**

Question 70

How can conditional genetics be used to activate and repress gene expression?

Answer 70

For conditional activation / repression: **mutated Cas9** (**dCas** - 'dead') - dCas9 with **fused activation** /**supression machinery** - **when** activator/supressor **added** - induces **activation** / **supression** machinery action

Question 71

What technique is used in animal cloning?

Answer 71

**Somatic cell nuclear transfer** (SCNT): **metaphase II oocyte enucleated**- **adult** differentiated cell **nucleus transferred** into enucleated oocyte - **grown** in vitro to **blastocyst** - **transferred** into **mother** -> offspring However, very **inefficient** procedure - **chromatin structure** (epigenetic marks) in differentiated cells **different to embryonic** Nuclear donor cells: fibroblasts, T lymphocytes

Question 72

What are the application of somatic cell nuclear transfer?

Answer 72

- **Reproductive cloning** - generating genetically identical animals - **Therapeutic cloning** - generating ES cells/ tissue / organs for regenerative medicine (**genetically compatible organs** - no rejection)

Question 73

Compare two method used in generating ES cells?

Answer 73

Reprogramming by: - **IVF**: oocyte fertilized with sperm -> blastocyst -> **ES** cells taken from **epiblast** => **99% survive** to **generate ES** cells - **SCNT**: **oocyte enucleated** -> **somatic nucleus injected** -> **ES** cells from **epiblast** => **5% survive** to **generate ES** cells ---> due to **abnormal epigenetic regulation** (chromatin structure in differentiated cells different compared to embryo) - inheritance independent of DNA sequence

Question 74

Are maternal and paternal pronuclei different? What confirms it?

Answer 74

**Yes**, different In **pronuclear transfer** - if **paternal pronucleus X exchanged with maternal** pronucleus - **no** embryo **development** => maternal and paternal are **different** **pronuclei** - **both required** for normal development => **gene imprinting** in gametes

Question 75

What is parental gene imprinting?

Answer 75

Parental gene imprinting: **some genes activated / repressed depending on parental origin** -> **monoallelic uniparental expression** **Imprinting** occurs **in gametogenesis** exclusively in eggs / sperm -> **different** imprinting **marks** -> persist into the zygote Both maternal and paternal needed because **gene expression must be complementary**

Question 76

Explain how sex specific genomic imprinting occurs

Answer 76

1. Supression/activation **erasure in primordial germ cells** 2. **Imprint establishment** depending on embryo sex (birth-> puberty) 3. In reproduction both **gametes** with different imprints **form zygote** -> **blastocyst** 4. **Monoallelic expression** but **genetic imprinting compatibility** between males and females - expression of needed genes ensures **normal development**

Question 77

How are the imprinted genes organised?

Answer 77

Maternally/paternally imprinted genes are usually **clusterred** in chromosome regions - **imprinting controlled by same mechanisms in the region**

Question 78

How are imprinted genes studied?

Answer 78

Imprinted genes can be revealed **by mutation**: mutating gene which supposed ot be expressed **maternally**/**paternally** -> **gene not functional in both** (full mutation) Studying a gene on paternal X: if **abnormal** phenotype => **mutated** gene is **paternally expressed** if **normal** phenotype => **mutated** gene is **maternally expressed**

Question 79

What are the major roles of maternally/paternally expressed genes and what is the main mechanism of imprinting?

Answer 79

~**80** imprinted genes **identified** - **expected more** - have major effects on **fetal/placenta growth**, **postnatal behaviour** **Maternally** expressed genes - **supress fetal growth** **Paternally** expressed genes - **enhance fetal growth** DNA **methylation** - **main mechanism** but also epigenetic modifications also used

Question 80

How differentiated cells can be reprogrammed into pluripotent cells?

Answer 80

**iPSCs**: 24 TFs identified to **reverse differentiation** - only **4 TFs** needed for in-vitro de-differentiation: **Oct4**, **Sox2**, **Klf4**, **c-Myc** sufficient to turn mouse fibroblasts into iPSCs - use **retroviruses** to **infect embryonic fibroblasts** with the needed **de-differentiation genes** - re-differentiation **using specific signalling molecules**

Question 81

What is the potential of iPSCs for therapeutic use?

Answer 81

**iPSCs** can be generated **from patient's cells** and **re-differentiated** into needed cell type - **genetically compatible** no rejection

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