VL3 - Developmental Biology Flashcards
How does a single cell give rise to an entire embryo?
1. Fertilization (fusion of sperm and eggcell)
2. Cleavage (Zygote undergoes a series of rapid cell devision. The cells in Morula, called blastomers, continue to divide without increase in overall size)
3. Bastulation (cleavage leads to formation of blastocytes. A fluid-filled hollow sphere composed of an outer layer of cells called the throphoblast and an inner cell mass.)
4. Gastrulation (Blastocytes undergoes gastrulation, process druing which the three primary germ layers - ectoderm, endoderm, mesoderm are established)
5. Neurulation (Ectorderm undergoes neurolation, development of central nervous system)
6. Organogenesis (Germ layers diffrentiate and undergo organogenesis, forming the rudimentary stuctures of organs and tissue.
7. Fetal Development (Embryo transizions into fetal stage, where further grow, maturation and specialiaziation of tissue and organs occure.
Development of the embryo from fertilized egg is known as embryogeneis
How are diffrent cell types determined?
1. Mosaic Development:
- unequal distribution of cytoplasmic determinants in the egg, leading to predetermined cell fates based on the positional information with the embryo
- cell fate is largely set early in development
- maternal substances in specific regions of the egg contribute to cell fate determination
cells in diffrent regions follow distinct development pathways
2. Regulative Development:
- flexible cell fate dtermination, where cells can adjust their fate based on interactions with neighbouring cells and external signals
- Cell fate is not fixed early on - becomes determined through cell-cell signaling and interactions
- cell can adjust their fate based on extrinsic signals and the embryonic environment
- Greater platicity in cell fate determination
–> Interplay between both systems: Many organisms and developmental processes exhibit characteristics of both mosaic and regulative development.
How is a three-dimensional body built?
Throughout embryogenesis, the interplay of genetic information, cell signaling, and environmental cues ensures the precise and coordinated development of a three-dimensional body with diverse structures and functions.Cellular behaviors control the morphogenesis of tissue and organs.
Three types of developmenta biology experiments
1. Observation: Correlative evidence –> Find it
2. Loss-of-function(LOF): evidence to establish necessity –> Loose it
3. Gain-of-function (GOF): evidence to establish sufficiency –> Move it
–> They must consistently support each other to establish and solidify a conclusion
What are the major tools used in alle animal model systems?
- Mutagenesis
- CRISPR/Cas9
- Transgenesis methods
- Genetic recombination systems (Cre-locP, Flp-FRT)
- Genetic driver system (UAS/Gals4)
Mutagenesis strategies
1. Genom-wide mutagenesis (foward genetics)
- The whole genome is mutagenized to identify genes that are involved in specific process
- starting from random single-gene mutant
- genomic analysis at the DNA level
- check for single-gene inheritance patterns
- validation of gene function and mutant phenotypes
–> An unbiased way to identify genes that function in a particular process, whereas the mutants themselves are very valuable resources for dissecting the function of the gene
Note: not all animal models are suitable for foward genetic screens. Requirement is easy screening through easy breeding, a high number of progency and tractability of phenotypes
Foward genetics: phenotype –> gene (mapping of a mutation)
2. Gene-target mutagenesis (reverse genetics)
- A target gene is specifically mutagenized
- start with known molecule and then attempts to disturb this molecule to acess the role of the normal gene product
- dirturb molecules with transgenesis, CRISPR/CAS9 genome editing technology, Morpholinos, RNAi
Reverse genetics: gene –> phenotype
- Transgenic techology: transgenic lines to overexpress a normal modified gene
- Gene silencing: knocking-out (deleting a gene whe and where you want)
Mutagenesis = Destroy/Modify gene –> observe phenotype and infer function of gene
Reverse genetics
starts with a known 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 is CRISPR/Cas9 ?
Reverse genetics on DNA Level
A bacterial adaptive defence/immune system against viruses and plasmids
Nobelprize in Chemistry (2020) to Emmanuella Charpentier and Jennifer Doudna for “the development of a method for genome
editing”
How mutations are introduced?
VL3 Folie 28 ??
A new generation with a ne version of Cas9 proteins, either egineered or from diffrent bacteria can target RNAs.
CRISPR = Clustered Regularly Interspaced Short Palindromic Repeats
Cas9 = CRISPR-associated protein 9 (nuclease)
PAM = Protospaceradjacent motive
Morpholinos
Reverse genetics on RNA Level
Morpholino = Short oligomer of about 25 bases, conatining nuclei acid analogues, that inhibit gene function. Composed of morpholine rings linked together by phosphorodiamate groups.
Commonly used as synthetic analogs of nuclei acids for applications such as: gene knockdown or gene expression modulation.
Note:Morpholine is a chemical compound with applications in chemistry and industry, while morpholino refers to a class of synthetic molecules used in molecular biology and genetics. Both contain morpholine rings.
RNA interfernece (RNAi)
Reverse genetics on RNA Level
RNA interference is a natural cellular process that involves the regulation of gene expression through the introduction of double-stranded mRNA by which mRNAs are targeted for degradation by double stranded RNA of identical sequences.
Powerful tool to controll the activity of genes and a common tool for knowcking down genes and downregulating or silencing gene expressions.
- Generation of small RNA Molecules
- Dicer Processing
- Loading onto RNA-Induced Silence Complex
- Target Recognition and Binding
- mRNA Degradation or Translation Inhibition
Transgenesis
Reverse genetic on DNA Level
Transgenesis:
Foreign DNA can be randomly insterted into genome and therfore disturpt gene function
By unsing: transposable elemnts (transposon = a DNA sequence that can excise and insert itself into diffrent positions of the genome)
Introduction of new or modified genetic material from one organism to another
Methods:
1. Target insertion: in a specific genome locus
e.g: CRISPR/Cas9 mediated knock in via homolgy independent mechanism
2. Non-target insertion: random insertion sites
e.g: transposable elements
CRISPR/Cas9 mediated knock in via homolgy independent mechanism
Transgenesis
1. Delivery of CRISPR/Cas9 complex into target cell through e.g microinjection, electrophoration or viral vectors.
2. Cas9 cleavage at target site.
The gRNA guides thes Cas9 Enzyme to the specific target DNA sequence. Cas9 induces DSB (Double strand break)
3. Homolgy-independet repair mechanism
Cells natural repair machinery is engaged to rpeair DSB.
4. Slection and screening
modiefied cells are selcted and screened for the successful intergration of the knock-in sequence.
Orientation can be controlled and random.
If orientation is controlled the DNA to be inserted is flanked by sequences that match the locus, they are homologous
Transposable elements
Transgenesis
Transposable element (transposon) is a DNA sequence that can exercise and insert itself into diffrent positions of the genome often reffered as “jumping gene” or “mobile genetic element”.
Transposons were first discovered in corn by Babara McClintock. (Nobeprize in 1983)
- DNA transposons
- Virus like retrotransposons
- Poly A retro transpososns
Gene expression systems are essential for controlling when, where and to what extent a particular gene is active or expressed within an organism.
- Promoter Strength: weak promoter in construct yields minimal expression.
- Enhancer Impact: Insertion near endogenous enhancer can enhance expression pattern.
- Insertion Site Significance: Study region around insertion to identify and study specific gene expression.
- Outcome: Pinpoint nearby genes involved in the observed expression pattern.
Gene expression systems
Transgenesis
- conditional and inducible
- control time and location
- control gene expressions in specific and inducible manner
–> Gal4/UAS system
–> FRT/FLP system
–> Cre-Lox System
Explain the Gal4/UAS system
Transgenesis
conditional expression system
- Gal4 = yeast TF (not naturally presented in organism of interest)
- UAS(Upstream activating sequence) = specific DNA sequence that GAL4 bind to
Example Fruitfly:
1. Gal 4 Fly Line –> Driver Line
This line contains a Gal4 gene which codes for a Gal4 Protein and a tissue specific promoter gene.
Allows Gal4 only to be produced in certain tissues.
If Promoter is active, Gal4 will be expressed.
- UAS Fly Line –> Reporter Line
Has a UAS Region, next to the gene that the researchers want to study called the reporter gene (e.g: GFP)
UAS is all over the fly but can only be activated by Gal4. - Crossing the 2 lines
- Creating a fly with Gal4 and UAS
So when the tissue specific promoter is active and allows Gal4 gene to express Gal4 Protein which binds to UAS. This triggers the expression of the gene that is attached to the UAS. (glowing green)
–> Tells researchers where the target gene is expressed in the fly
Explain the Flp/FRT System for site-specific recombinations
Transgenesis
conditional expression system for site-specific recombinations
similar to Cre-Lox
-
Flp (flippase):
recombinase Enzyme from yeast
FLP recognizes a pair of FRT Sequences which are flanking a genome of interest -
FRT (Flippase recognition target):
refers to specific DNA sequence recognized by FLP. FRT sites have a defined orientation and recombination occurs between sites with the same orientatin.
–> induction of site-specific recombination after DNA replication
–> mutagenized chromosome arm is
homozygous in clones of cells
Cre/Lox System
Transgenesis
Conditional expression system
that allows for the targeted deletion, inversion or translocation of DNA sequences.
- Cre recombinase: (CausesRecombination)
Enzyme derived from bacteriophage P1
A Cre-mouse contains Cre recombinase transgene and tissue specific Promoter - loxP site: (Locus of crossing (x) over, P1)
2 Lox-sites flank a sequence of interest = “foxed” locus
specific DNA Sequence recognized by Cre.
–>The orientation if locP sites determines the outcome of Cre-mediated recombination.
- usually there is a Cre-mouse and a Lox-mouse, which are developed independent from each other. These 2 Mice are crossed.
–> Cre moiuse can be designed to express cre recombinase only under certain conditions
(Many diffrenrt Cre -mediated model system can be constructed)
Deletion: when 2 loxP sites are in the same orientation –> Cre catalyzes the deletion of the DNA segment between
Inversion: If loxP sites are in the opposite orientations, Cre can cause an inversion/flip of the DNA segment
Translocation
If the loxP sites are located on different strands of DNA and are oriented in the same direction, Cre recombinase mediates a translocation of the segment.
Applications:
- conditional gene knockout
- cell-type specific modification
