Correlating endogenous gene and reporter gene knockdowns

Occasionally a nice fundamental paper comes along and the new paper from Kamachi et al. is just such a treat.

Morpholinos are widely used to block pre-mRNA splicing and mRNA translation. Back in 2001, McKeon et al. described the dose-response of splice-modifying Morpholinos using a luciferase up-regulation system. Now in 2008, Kamachi et al. have explored knockdown efficacy of translation-blocking Morpholinos using a luciferase down-regulation system. In addition to measuring knockdown efficacy of several different oligos targeting the same 5'-UTR, they varied the doses, tried coinjection of oligo combinations (showing synergistic response), and tried the oligos against various mispaired targets.

The following two citations are useful background reading for anyone starting to work with Morpholino antisense and are good references to fundamental quantitative measurement of Morpholino activity.

New paper:
Kamachi Y, Okuda Y, Kondoh H. Quantitative assessment of the knockdown efficiency of morpholino antisense oligonucleotides in zebrafish embryos using a luciferase assay. Genesis. 2008 Jan 14;46(1):1-7 [Epub ahead of print]

Oldie but goodie:
McKeon J, Cho MJ, Khaledi MG. Quantitation of Intracellular Concentration of a Delivered Morpholino Oligomer by Capillary Electrophoresis-Laser- Induced Fluorescence: Correlation with Upregulation of Luciferase Gene Expression. Anal Biochem 2001 Jun 1;293(1):1-7