8.) Genome Modification, Northern and Western Blots To further investigate the f

Question

8.) Genome Modification, Northern and Western Blots To further investigate the function of CREB you decide to create a CREB-KO cell line. To this end you design a Cas9 system to create a doublestrand break in the middle of CREB exon 2 (indicate by lightning) that you expect will be fixed imperfectly by the cellular Non-Homologous End-Joining (NHEJ) DNA repair machinery. To check for the success of Cas9 treatment you collect RNA and Protein from your cell line before and after Cas9 treatment and run Northern and Western Blots using probes and antibodies binding CREB mRNA and protein as indicated below Cas9 CREB Exon 2 DNA Doublestrand Break Probe 1 Probe 2 CREB ExonmRNA 3-UACACCGUUAGACACC-5 3-GUCGUCGUAGAGGUGA-5 5-GGGCUAAUGUGGCAAUCUGUGGCUGGGCUUGAACUGUCAUUUGUUGAUUUUCAGCUUCUGUUACAGCAGCAUCUCCACU-3 CREB Exon 2 AA E SG A D NOS GD A AV TE Antibody1 Antibody 2 Western Blots Northern Blots Probe 1 A.) Northern Blots: What does the banding pattern produced by the two probes tell you about the properties of CREB mRNA before and after Cas9 treatment? (Word limit: 40) (6 points) Probe 2 Antibody 1 Antibody 2 Before After Before After Cas9 Cas9 Cas9 Cas Before After Before After Cas9 Cas9 Cas9 Cas9 70 60 50 40 30 20 10 2000 B.) Western Blots: What does the banding pattern produced by the two antibodies tell you about the properties of CREB protein before and after Cas9 treatment (Word limit: 60) (12 points) C.) By combining the conclusions drawn from Northern and Western Blots, explain what kind of mutation the Cas9 mediated doublestrand break most likely created in CREB exon 2 and what the consequences of this mutation are. (Word limit: 40) (6 points)

Explanation / Answer

It is possibility that the repair that carried out by homologous recombination contain defective gene copy where due to mutation intron one will not spliced out. Hence it comes in transcription and give the slightly higher band than the before treated mRNA.

If check the western blot that show that as first antibody bind to protein seuqnce and changes or intron I, between exon I and exon II, transcription may incorporate premature termination of of protein formation hence second antibody is unable to work.

If we combine the results than can say homologous recombination add some extra bases or that change the splicing pattern of Intron I. Intron may contain sequence that leads to premature termination. Hence the signal of both the probe is slightly higher whereas protein is prematurely terminated.

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