Analyze The Data

RNA interference (RNAi) is a process of post-transcriptional gene silencing mediated by short double-stranded RNA molecules called siRNA (small interfering RNAs). In mammalian cells, transfection of 21-22 nucleotide siRNAs leads to degradation of mRNA molecules that contain the same sequence as the siRNA. In the following experiment, siRNA and knockout mice are used to investigate two related cell surface proteins designated p24 and p25 that are suspected to be cellular receptors for the uptake of a newly isolated virus.

a. To test the efficacy of RNAi in cells, siRNAs specific to cell surface proteins p24 (siRNA-p24) and p25 (siRNA-p25) are transfected individually into cultured mouse cells. RNA is extracted from these transfected cells and the mRNA for proteins p24 and p25 are detected on Northern blots using labeled p24 cDNA or p25 cDNA as probes. The control for this experiment is a mock transfection with no siRNA. What do you conclude from this Northern blot about the specificity of the siRNAs for their target mRNAs?

Probe = p24 cDNA

Probe = p24 cDNA

Control siRNA-p24 siRNA-p25

Probe = p25 cDNA

Probe = p25 cDNA

Control siRNA-p24 siRNA-p25

b. Next, the ability of siRNAs to inhibit viral replication is investigated. Cells are transfected with siRNA-p24 or siRNA-p25 or with siRNA to an essential viral protein. Twenty hours later, transfected cells are infected with the virus. After a further incubation period, the cells are collected and lysed. The number of viruses produced by each culture is shown below. The control is a mock transfection with no siRNA. What do you conclude about the role of p24 and p25 in the uptake of the virus? Why might the siRNA to the viral protein be more effective than siRNA to the receptors in reducing the number of viruses?

Cell Treatment

Control siRNA-p24

siRNA-p25

siRNA-p24 and siRNA-p25 siRNA to viral protein

Number of Viruses/ml

c. To investigate the role of proteins p24 and p25 for viral replication in live mice, transgenic mice that lack genes for p24 or p25 are generated. The loxP-Cre conditional knockout system is used to selectively delete the genes in cells of either the liver or the lung. Wild type and knockout mice are infected with virus. After a 24-hour incubation period, mice are killed and lung and liver tissues are removed and examined for the presence (infected) or absence (normal) of virus by immunohistochemistry. What do these data indicate about the cellular requirements for viral infection in different tissues?

Tissue Examined

Mouse

Liver

Lung

Wild type

infected

infected

Knockout of p24 in liver

normal

infected

Knockout of p24 in lung

infected

infected

Knockout of p25 in liver

infected

infected

Knockout of p25 in lung

infected

normal

d. By performing Northern blots on different tissues from wild-type mice, you find that p24 is expressed in the liver but not in the lung, whereas p25 is expressed in the lung but not the liver. Based on all the data you have collected, propose a model to explain which protein(s) are involved in the virus entry into liver and lung cells? Would you predict that the cultered mouse cells used in parts (a) and (b) express p24, p25, or both proteins?

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