Find It, Lose It, Move It Case Study

finding the mRNA or the protein and where it is expressed

what is the process of characterizing gene expression

1) generate RNA complementary to gene of interest and conjugate it with DIG protein

2) place DIG-conjugated probe in organism, so it will bind to mRNA of gene of interest

3) antibody binds to DIG and has alkaline phosphatase enzyme attached to it

4) enzyme cleaves molecule, generating blue color that can be visualized

explain the process of in-situ hybridization

1) take all of the RNAs from a chunk of tissue

2) use reverse transcriptase to convert the RNAs into cDNAs

3) amplify the cDNA and sequence it

4) can now process and map to the genome to see what is being expressed

explain process of RNA-seq

to see how gene expression of a tissue changes over developmental time because it determines all of the RNA being expressed in a tissue

what is RNA-seq commonly used for

1) create primary and secondary antibodies against antigens on a protein

2) on secondary antibody, a substrate-enzyme reaction occurs that results in color to be produced

3)

explain process of immunohistochemistry (IHC)

direct → primary antibody directly has a fluorophore on it, so we can see where it is

indirect → primary antibody with secondary antibody bound to it which has a fluorophore on it that can be visualized

explain immunofluorescence

labeling proteins within a tissue

what is IHC and IF used for

Cas9 → endonuclease that cuts DNA and gives double stranded break

sgRNA → RNA made that has a sequence complementary to wherever we want to cut the genome → must be adjacent to PAM which show up commonly

what are the 2 components required for CRISPR

1) generate sgRNA complementary to region of interest so it can bind

2) sgRNA then recruits Cas 9 which cuts the DNA to give ds break

3) nonhomologous end joining or homology directed repair

explain process of CRISPR

DNA repair enzymes want to fix broken DNA, so they will try, but it’s not perfect → leads to insertions or deletions that cause frame shift mutations and premature stop codons that lead to truncated or nonfunctional proteins

nonhomologous end joining in CRISPR

insert DNA fragment in which the ends match the opposite strand of DNA so it base pairs → DNA repaired and has another fragment such as a portion of interest

homology directed repair in CRISPR

1) IHC/ICC/IF

2) in situ hybridization

3) RNA-seq

4) fluorescently tagged proteins through CRISPR

Find it techniques to use

1) CRISPR → nonhomologous end joining

2) miRNAs/RNAi

3) pharmacological inhibitors

4) function blocking antibodies

Lose it techniques