2. VL

Question 1

How does a single fertilized egg cell give rise to an entire mamalin embryo? (circle)

Answer 1

FERTELIZED EGG – Cleavage –>
BLASTOCYST – Implantation –>
in UTERINE WALL –Gastrulation and Turning –> –>Orgaogenesis –> fetal growth and development –> Birth –> ADULT

Question 2

what are the different cell types in a zebra fish in an early gastrula stage?

Answer 2

ectoderm (epidermis), yolk cell, neural ectoderm, mesoderm somites, mesoderm (blood forming), notochord, endoderm and mesoderm (mesendoderm)
fertilized egg gots more and more differentiated

Question 3

What are some major tools, used in all (!) animal model systems?

Answer 3

Mutagenesis (classical and molecular biology methods)
CRISPR/Cas9 genome editing
Transgenesis methods
Genetic recombination systems (Cre- loxP; Flp-FRT) Genetic driver systems (UAS/Gal4)

Question 4

What underlies the organization of multiple cell types into tissues and organs?
big questions of developmental biology can be solved by…

Answer 4

…model organisms

Question 5

principle of mutagenesis?

Answer 5

destroy a gene, observe phenotype and infer function of gene

Question 6

mutations (dublication, point mutation, deletion rate) in nature (Drosophila melongaster or C. elegans) caused by…

Answer 6

exogenous environmental insult, endogenous oxidative damage, repair errors

Question 7

what are the two mutagenesis strategies? (3)

Answer 7

1. Genome-wide mutagenesis(forward genetics)
   • The whole genome is mutagenized to identify genes that are involved in a specific process, e.g. developmental.
2. Trangenesis-mediated mutagenesis (transposable elements)
3. Gene-targeted mutagenesis(reverse genetics)
   • A target gene is specifically mutagenized.
   • CRISPR/Cas9 genome editing

Question 8

one can understand biological property by discovering the genes that controls it - how?

Answer 8

• starting from random single-gene mutants
• genomic analysis at the DNA level
• check for single-gene inheritance patterns
• validation of gene function and mutant phenotype

Question 9

which model animals are suitable for forward genetic screens?

Answer 9

•easy screening through easy breeding
•high number of progeny
•tractability of phenotypes
–> C. elegans, Drosophila, no mouse

Question 10

If you use a chemical agent for mutagenesis, what chemical can you use and what effect can you get?

Answer 10

Ethylnitrosourea (ENU) - Mostly point mutations
Ethyl methanesulphonate (EMS) - mostly point mutations
Trimethylpsoralen - Small deletions (100 bp to 15 kb)

Question 11

what mutagenesis can you get using X-ray or gamma rays?

Answer 11

Large genomic deletions or translocations (Ionizing radiation)

Question 12

Reverse genetics starts with a know molecule (DNA sequence, mRNA or protein) and then attempts to disrupt this molecule to assess the role of the normal gene product in the biology of the organism. What are examples?

Answer 12

• CRISPR/Cas9 genome editing technology
• ZFNs
• TALENs
• Recombination-mediated site-directed mutagenesis
• RNAi
• Morpholino

Question 13

What are the advantages to use CRISPER?

Answer 13

• easy to use
• cheap
• works in all animal models
• adaptable

Question 14

What means CRISPER Cas?

Answer 14

clustered regularly interspaced short palindromic repeats (CRISPR)
cas genes (CRISPR- associated)

Question 15

What do you need for genom editing ?

Answer 15

• Cas9 protein
• gRNA
• PAM (protospacer adjacent motive)

Question 16

What can you do with CRISPER Cas?

Answer 16

genom activation and repression
orthogonal activation and repression with scRNA
epigenetic modification

Question 17

What is transgenesis and what can you use it for?

Answer 17

• introduction of new or modified genetic material into eukaryotic cells
• central technology to basic genetic research
–> Genetically modified organisms (GMO) in S1 labs or higher

Question 18

transgegesis methodes? (5)

Answer 18

transposable elements
homology-directed recombination
CRISPR/Cas9 mediated genome engineering
viral infection
transfection

Question 19

what is a transposable element (transposon)?

Answer 19

A DNA sequence that can excise and insert itself into different positions of the genome.

Question 20

in which animals can you use microinjections?

Answer 20

zebrafish, mouse, drosophila

Question 21

Which conditional and inducible gene expression systems do you know?

Answer 21

• GAL4/UAS system
• FRT/FLP system
• Cre-Lox system
–> control of location and time

Question 22

How does conditional expression system: GAL4/UAS works?

Answer 22

• can express certain cloned genes in specific cells or tissues.
• GAL4 = yeast-specific transcription factor under the control of a weak promoter, for expression, another promoter or enhancer is needed
• protein GAL4 specifically binds to the so-called UAS (upstream activating sequence), thus activating a downstream target gene(GFP)

Question 23

how does conditional expression system: Flp/FRT system for site-specific recombinations works? (somatic mosaics in eyes)

Answer 23

Flp (flippase) recombinase from yeast 2μ plasmid; under expression control (e.g. heat-shock promotor)
• FRT (Flippase recognition target) sites at identical position on homologous chromosomes
• ! mutated gene !
induction of site-specific recombination after DNA replication!
mutagenized chromosome arm is homozygous in clones of cells

Question 24

how does conditional expression system: Cre/Lox system works?

Answer 24

A: excision: cis replacement of LOX p-sites in same directional orientation
B: inversion: cis replacement of LOX p-sites in opposite directional orientation
C: translocation: trans replacement of LOX p-sites

• derived from P1 bacteriophage
• Cre recombinase recognises 34 bp loxP sites
• orientation and location of loxP sites determines recombination outcome

Question 25

what are the key concepts in studying the role of genes?

Answer 25

• destroy it
• change it
• express it in a spatially and temporally controlled manner
with: mutagenesis, transgenesis, genome-editing