2. VL Flashcards
Methodology
How does a single fertilized egg cell give rise to an entire mamalin embryo? (circle)
FERTELIZED EGG – Cleavage –>
BLASTOCYST – Implantation –>
in UTERINE WALL –Gastrulation and Turning –> –>Orgaogenesis –> fetal growth and development –> Birth –> ADULT
what are the different cell types in a zebra fish in an early gastrula stage?
ectoderm (epidermis), yolk cell, neural ectoderm, mesoderm somites, mesoderm (blood forming), notochord, endoderm and mesoderm (mesendoderm)
fertilized egg gots more and more differentiated
What are some major tools, used in all (!) animal model systems?
Mutagenesis (classical and molecular biology methods)
CRISPR/Cas9 genome editing
Transgenesis methods
Genetic recombination systems (Cre- loxP; Flp-FRT) Genetic driver systems (UAS/Gal4)
What underlies the organization of multiple cell types into tissues and organs?
big questions of developmental biology can be solved by…
…model organisms
principle of mutagenesis?
destroy a gene, observe phenotype and infer function of gene
mutations (dublication, point mutation, deletion rate) in nature (Drosophila melongaster or C. elegans) caused by…
exogenous environmental insult, endogenous oxidative damage, repair errors
what are the two mutagenesis strategies? (3)
- Genome-wide mutagenesis(forward genetics)
• The whole genome is mutagenized to identify genes that are involved in a specific process, e.g. developmental. - Trangenesis-mediated mutagenesis (transposable elements)
- Gene-targeted mutagenesis(reverse genetics)
• A target gene is specifically mutagenized.
• CRISPR/Cas9 genome editing
one can understand biological property by discovering the genes that controls it - how?
- 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
which model animals are suitable for forward genetic screens?
•easy screening through easy breeding
•high number of progeny
•tractability of phenotypes
–> C. elegans, Drosophila, no mouse
If you use a chemical agent for mutagenesis, what chemical can you use and what effect can you get?
Ethylnitrosourea (ENU) - Mostly point mutations Ethyl methanesulphonate (EMS) - mostly point mutations Trimethylpsoralen - Small deletions (100 bp to 15 kb)
what mutagenesis can you get using X-ray or gamma rays?
Large genomic deletions or translocations (Ionizing radiation)
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?
- CRISPR/Cas9 genome editing technology
- ZFNs
- TALENs
- Recombination-mediated site-directed mutagenesis
- RNAi
- Morpholino
What are the advantages to use CRISPER?
- easy to use
- cheap
- works in all animal models
- adaptable
What means CRISPER Cas?
clustered regularly interspaced short palindromic repeats (CRISPR) cas genes (CRISPR- associated)
What do you need for genom editing ?
- Cas9 protein
- gRNA
- PAM (protospacer adjacent motive)
What can you do with CRISPER Cas?
genom activation and repression
orthogonal activation and repression with scRNA
epigenetic modification
What is transgenesis and what can you use it for?
• 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
transgegesis methodes? (5)
transposable elements homology-directed recombination CRISPR/Cas9 mediated genome engineering viral infection transfection
what is a transposable element (transposon)?
A DNA sequence that can excise and insert itself into different positions of the genome.
in which animals can you use microinjections?
zebrafish, mouse, drosophila
Which conditional and inducible gene expression systems do you know?
• GAL4/UAS system
• FRT/FLP system
• Cre-Lox system
–> control of location and time
How does conditional expression system: GAL4/UAS works?
- 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)
how does conditional expression system: Flp/FRT system for site-specific recombinations works? (somatic mosaics in eyes)
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
how does conditional expression system: Cre/Lox system works?
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
what are the key concepts in studying the role of genes?
• destroy it
• change it
• express it in a spatially and temporally controlled manner
with: mutagenesis, transgenesis, genome-editing
