B Pyrowolakis Drosophila

recessive: phenotype only when homozygous, in heterozygous mutants the dominant allel is observed -> Example: white eye

dominant: phenotype even in heterozygous mutants -> Ex: curly wing

null allel: loss-of-function mutation where the gene completely loses its function. This can be due to deletions, nonsense mutations, or mutations that severely disrupt the gene's structure

hypomorphic: loss-of-function mutation where the gene product has reduced activity compared to the wild type

neomorphic: gain-of-function mutation where the gene acquires a new function that is not found in the wild type -> Ex: antennapedia

regulatory: affects the expression levels, timing, or location of a gene without altering the gene product itself. Mutation in regulatory regions like promoters, enhancers

conditional allele: displays a phenotype only under certain environmental conditions -> Ex: temperature-sensitive alleles

GoF: overexpressing, activating or enhancing functions of a specific gene -> Ex: antennapedia

also a transplantation experiment could be seen as a gain of function

LoF: gene knock out or knock down, also conditional knock outs very important

Substances with organizer properties

Form concentration/activity gradients

Directly instruct cell fates

Instruct cell fates in a dose dependent manner [THRESHOLD]

Long Range action

Stability and Diffuseibility

Organizer properties: with a GoF or Lof experiment and observe if organization is disrupted

Gradient / diffusability / long range: use of reporter constructs e.g. GFP linked to the morphogen

Directly instructs cell fate: Induce the morphogens signal at a different position see if the same direct effect happens

Threshold / dose dependent manner: also with reporter constructs, observe the different concentrations and compare the cells fates if the cells at lower conc of the gradients have different fates it is threshold dependent

Distribution: ability of a signaling molecule, such as a morphogen, to spread out over a considerable distance from its source

Function: ability of a signaling molecule to elicit biological responses in cells located far from its source

• translational reporter using e.g. GFP

• GFP fused to protein of interest at N- or C-terminal

• allows visualization of protein distribution and gradient formation

• a protein/ morphogen gradient is instructive when the protein is able to activate downstream signaling in cells along the gradient even at low concentrations

• antibody staining for signaling components that are activated by signaling pathways, epitopes have to be chosen that are exclusively present in active signal transuction (phosphoMad in dpp signaling)

• mosaic mutations: not present in all cells, usually arise from somatic mutations

• mosaic genetic analysis = clonal screen?

• analysis of mutations that are not present in every cell of an organism e.g. only in head. allows analysis of otherwise embryonic lethal mutations and investigation of which tissues mutant cells fail to contribute to.

• chimeras, conditional alleles

Introducing a Plasmid containing the gene of interest by serveral methaods (transfection, viral transduction, Crispr, Homologous rekombination, electroporation)

Selection of positive cells (AB resistance)

Cloning with cell sorting markers (fluoresence)

Screening for the protein (western blot) and continue culture in selective media

target vector has to contain promoter, protein of interest and selection cassette —> incorporatin into genome as transgene

monoclonal: single cells after transfection to make sure all cells arise from one precursor

Crispr-Cas, Zink Finger Nucleases or TALENS

guide RNA, or recognition sequences (VDR or Zinc Fingers) have to recognize gene of interest, induce double strand break in target gene

—> repair via homologous recombination, introduction of donor vector that carries promoter, mutated poi and selection cassette necessary to have prooer template

—> introduction of genome editing tools via transfection?

clonal analysis depends on usage of conditional alleles e.g.: Cre and FLP recombinases. The expression of the recombinases can be controlled in a temporal and spatial manner.

• clone position —> recombinase driven under tissue-specific promoter

• clone size —> control of how long recombinase is expressed e.g. via CreER (duration of tamoxifen treatment) or TetON/TetOFF (duration of dox treatment)

• clone number —> mosaic clones, introduction of transgenes into es cells, then microinjection into blastocyst, usage of chimera?

dpp = anterior-posterior patterning of wing axis, dorso-ventral axis of embryo

wingless = loss of function resulzs in loss of wings

Notch = results in notched wings

hedgehog = trichome formation

snail = mucus production ?

ken and barbie = no external genitalia

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