Let’s say that you discover a new species of insect
a. You use whole mount in situ hybridization (WISH) to show that protein A is present only in the dorsal half of the embryo. What are two other techniques to confirm localization of protein A, and how is each different from WISH?
b. You use a morpholino to target protein A as you test the hypothesis that protein A prevents protein B (a transcription factor) from entering the nucleus where it activates the expression of genes required to specify cells as ventral. What is the expected phenotype, and what are two important controls in this experiment?
c. You use CRISPR to disrupt the expression of protein C (a transcription factor) as you test the hypothesis that protein C inhibits endodermal genes when activated by protein B. What is the expected phenotype, and how could you disrupt the expression of a different gene to produce the same phenotype?
a. The two other techniques are -
Immuno-fluoresence (IF) studies for colocalisation of specific protein and
Immuno-precipitation (IP) technique for protein- protein association .
IF techniques is used for identification and co-localisation of particular protein may be present in cytosolic or nuclear protein under the fluoresence microsopy.
IP technique is anlysed for isolation and other protein interaction of others protein or transcription factor using antibody stainning.
b. Positive control and negetive control.
In positive control, inputs (target protein A) are given with other molecule and detected by antidody stanin in western blot analysis.
negative control only interacting protein B without target protein. so no protein- protein interaction and no band found in western bloting technique.
c. Protein-C inhibits the endodermal gene and protein-B activated the protein-C .If Protein-C is disrupted by CRISPR , the endodermal gene expresses as normaly with proper function and expected phenotype.
Using the inhibitory RNA (RNAi) technique i.e short hairpin-RNA , micro-RNA etc. we can disrupt the expression of different genes. in this techniques RNA is inhibited and function protein is not produced . So the protein expression is disrupted.
Let’s say that you discover a new species of insect a. You use whole mount in...
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