Chrono Knockdown Using shRNA

Seven shRNAs targeting different regions of Chrono gene were designed. A nonspecific (NS) shRNA construct was used as a control. Synthetic oligonucleotides were annealed and cloned into pENTR/U6 (Invitrogen) and subsequently cloned into the pLL3.7GW vector as previously described [27] (link). The NIH 3T3 reporter cells were then infected with shRNA viruses.

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Anafi R.C., Lee Y., Sato T.K., Venkataraman A., Ramanathan C., Kavakli I.H., Hughes M.E., Baggs J.E., Growe J., Liu A.C., Kim J, & Hogenesch J.B. (2014). Machine Learning Helps Identify CHRONO as a Circadian Clock Component. PLoS Biology, 12(4), e1001840.

Publication 2014

pENTR/U6

Gene Nih 3t3 cells Oligonucleotides Shrna Vector Viruses

Corresponding Organization : Morehouse School of Medicine

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Variable analysis

independent variables

ShRNA constructs targeting different regions of the Chrono gene
Nonspecific (NS) shRNA construct

dependent variables

Infection of NIH 3T3 reporter cells with shRNA viruses

control variables

Transfection method (infection with shRNA viruses)

controls

Positive control: Nonspecific (NS) shRNA construct
Negative control: Not explicitly mentioned

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