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23 May 2018

♣ Explain the process of RNAi and how it contributes to differential gene expression

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Beverley Smith
Beverley SmithLv2
23 May 2018

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Related questions

a) You wish to investigate a gene's function in C. elegans. Describe one advantage and one disadvantage of using RNAi vs. gene deletion to investigate your gene of interest.

b) Briefly describe how double-stranded RNAs are delivered to C. elegans in order to perform RNAi.

In a genetic experiment:

The use and limits of RNA interference as a reverse genetics technique for knocking down gene expression were tested. The wild-type and mutants worms for the strains were selected. The RNAi feeding strains and control bacterial were used for the the RNAi experiment. The eri-1 C. elegan strains (wild-type) were used for RNAi treatment and mutant strains that were used ( unc-22 and dpy-13) C. elegans. Both carbencillin and IPTG wered added to the RNAi feeding plates and E.coli containing the RNAi transgenes were spread on the plates. The antibodic carbacillin only allows growth of bacteria which contains the RNAI construct. IPTG induces the expression of the polymerase which then transcribes the siRNA in the bacteria. 6 NGM plates without bacteria were used and 20ul of 12.5 mg/ml carbencillin concentration were spread on the 6 of the plates. The carbencillin dried and then IPTG was spread (25ug/ml) on each plate. Then 6 plates with the RNAi bacterial strains were used. Theree of plates for strain one and the other three for strain the second strain. When IPTG dried , 150 ul of the appropraite bacteria was added onto each plate. 2NGM plates (without carbenicillin or IPTG) was spread with 150 ul of OP50 bacterial culture. OP50 is the regular E. coli strain for culturing C. elegans and does not contain the RNAi constructs. These plate were used as the control plates6. Then the plates were incubated for two days. Then the adult hermaphrodites from the RNAi feeding plates and the op50 plates were removed. Then from the eri-1 wildtype stock, 2 or 3 L4 hermaphrodites or
young adults were transferred onto each RNAi feeding plate. Then L4 worms were transferred to an intermediate plate first. After they have a minute to move away from each other and the eggs, and transfered to the RNAi plates. On the two control plates seeded with OP50, 2 L4 or young adult hermaphrodites were placed there. Plates were obtain as the reference mutants (unc-22, bli-1 or unc-13). Then the plates were incubated to allow the worm to develop and lay eggs in two days. Then the adult hermaphrodites from your RNAi feeding plates and the op50 plates were removed and then placed back into the incubator.


The op500 was the negative control plate wtih 80 and 300 progeny and unc-22 had around 50. (reference plates)

Mating in the RNAi plates

Dpy (60) x wildtype (15) and unc( 70) x wildtype ( 8) progeny

the phenotype was movement and it seemed similar phenotype within the progeny.

Can you help me understand the results and what is happening genetically in the experiment ? Are the for dyp-15 x unc-22 mating cross and unc-22 x eri-1 wildtype C. elegans. Based on the data:

Briefly discuss the results of the RNAi experiment. How closely did the RNAi-treated worms phenocopy the mutant worms?

Was the gene knockdown 100% effective?

Was the RNAi treatmentequally effective for the two genes? If not, discuss this result and why that could occured.

Please be specific and detailed

Genetics HELP ASAP

Genetics HELP ASAP In a genetic experiment: The use and limits of RNA interference as a reverse genetics technique for knocking down gene expression were tested. The wild-type and mutants worms for the strains were selected. The RNAi feeding strains and control bacterial were used for the the RNAi experiment. The eri-1 C. elegan strains (wild-type) were used for RNAi treatment and mutant strains that were used ( unc-22 and dpy-13) C. elegans. Both carbencillin and IPTG wered added to the RNAi feeding plates and E.coli containing the RNAi transgenes were spread on the plates. The antibodic carbacillin only allows growth of bacteria which contains the RNAI construct. IPTG induces the expression of the polymerase which then transcribes the siRNA in the bacteria. 6 NGM plates without bacteria were used and 20ul of 12.5 mg/ml carbencillin concentration were spread on the 6 of the plates. The carbencillin dried and then IPTG was spread (25ug/ml) on each plate. Then 6 plates with the RNAi bacterial strains were used. Theree of plates for strain one and the other three for strain the second strain. When IPTG dried , 150 ul of the appropraite bacteria was added onto each plate. 2NGM plates (without carbenicillin or IPTG) was spread with 150 ul of OP50 bacterial culture. OP50 is the regular E. coli strain for culturing C. elegans and does not contain the RNAi constructs. These plate were used as the control plates6. Then the plates were incubated for two days. Then the adult hermaphrodites from the RNAi feeding plates and the op50 plates were removed. Then from the eri-1 wildtype stock, 2 or 3 L4 hermaphrodites or young adults were transferred onto each RNAi feeding plate. Then L4 worms were transferred to an intermediate plate first. After they have a minute to move away from each other and the eggs, and transfered to the RNAi plates. On the two control plates seeded with OP50, 2 L4 or young adult hermaphrodites were placed there. Plates were obtain as the reference mutants (unc-22, bli-1 or unc-13). Then the plates were incubated to allow the worm to develop and lay eggs in two days. Then the adult hermaphrodites from your RNAi feeding plates and the op50 plates were removed and then placed back into the incubator. The op500 was the negative control plate wtih 80 and 300 progeny and unc-22 had around 50. (reference plates) Mating in the RNAi plates Dpy (60) x wildtype (15) and unc( 70) x wildtype ( 8) progeny the phenotype was movement and it seemed similar phenotype within the progeny. Can you help me understand the results and what is happening genetically in the experiment ? Are the for dyp-15 x unc-22 mating cross and unc-22 x eri-1 wildtype C. elegans. Based on the data: Briefly discuss the results of the RNAi experiment. How closely did the RNAi-treated worms phenocopy the mutant worms? Was the gene knockdown 100% effective? Was the RNAi treatmentequally effective for the two genes? If not, discuss this result and why that could occured. Please be specific and detailed

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