Biology Reference
In-Depth Information
R. Transformation
Transgenic C. elegans strains can be obtained by either microinjection (
Mello and
Fire, 1995; Mello et al., 1991
) or ballistic transformation (
Wilm et al., 1999
). For
microinjection, a coinjection marker, like rol-6(su-1006) (
Kramer et al., 1990
), can
be used to select the transformed animals. For ballistic transformation, a copy of the
wild-type unc-119 gene is included in the DNA construct as a selection marker when
the unc-119(ed3) mutant strain is used for transformation (
Praitis et al., 2001
). The
recent development of the Mos1 mediated insertion technique for integrating single
copy transgenes into a specific chromosomal location can also be used for generat-
ing 3
0
UTR reporter strains (
Frokjaer-Jensen et al., 2008
).
Links
For EST, mRNA and miRNA registries
WormBase:
http://www.wormbase.org
miRBase:
http://microrna.sanger.ac.uk
For vectors
Fire Lab C. elegans Vector Kit:
http://www.addgene.org/Andrew_Fire
or
ftp://ftp.
wormbase.org/pub/wormbase/datasets/fire_vectors
For miRNA target prediction algorithms and databases
PicTar:
http://pictar.mdc-berlin.de/
TargetScan:
http://www.targetscan.org/worm_12/
For mRNA 5
0
and 3
0
end annotations based on RNA-seq and ALG-1 binding sites
based on CLIP-seq
III. Summary
mRNAs are essential regulators of many basic cellular processes. As such, slight
deviations in the amount, timing, or location of miRNA expression can have large
effects on cell and organismal growth. The analysis of primary, precursor, and mature
miRNA levels as well as the identification and characterization of miRNA targets is
thus crucial for determining the step in miRNA biogenesis or function affected in a
particular mutant or disease. The methods described in this chapter provide a foun-
dation for analyzing these steps in the powerful model organism C. elegans.
Acknowledgments
The authors thank members of the Pasquinelli and Slack labs for their comments, critical reading of this
manuscript, and sharing of protocols. This work was supported by funding to A. E. Pasquinelli from NIH
(GM071654), and the Keck, Searle, V, Emerald, and Peter Gruber Foundations, and funding to F. J. Slack
from NIH (GM064701, AG033921) and the Ellison Medical Foundation. P. M. Van Wynsberghe was
supported by a Ruth L. Kirschstein National Research Service Award Number F32GM087004 from the
National Institute of General Medical Sciences.
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