11.3
Conditional knock down for specific gene by artificial microRNA (amiRNA)
Chaoyang Cheng, Tetsuya Kurata, and
Mitsuyasu Hasebe
Introduction
miRNAs are approximately 21 nt small RNAs
which derived from specific MIR gene loci. These miRNAs regulate a target gene
expression through mRNA degradation. Effects of artificially constructed miRNA
on knocking down a target gene have been examined in Arabidopsis and
Physcomitrella (Schwab et al., 2005, 2006; Khraiwesh et al., 2008).
In this chapter, we describe an inducible
knock-down system combining the advantage of amiRNAs and the inducible
expression method using XVE system (see chapter * in this manual).
References
Schwab
et al.,
Specific
effects of microRNAs on the plant transcriptome.
Dev
Cell 8: 517–527 (2005)
Schwab
et al.,
Highly
specific gene silencing by artificial microRNAs in Arabidopsis.
Plant
Cell 18: 1121–1133 (2006)
Khraiwesh
et al.,
Specific
Gene Silencing by Artificial MicroRNAs in Physcomitrella patens: An Alternative
to Targeted Gene Knockouts
Plant
Physiol. 148: 684-693 (2008)
Materials and Methods
Design for amiRNA
amiRNA sequences for specific genes were designed
using amiRNA designer web site (WMD2; http://wmd2.weigelworld.org/cgi-bin/mirnatools.pl?page=3). Criteria for selecting
amiRNA’s sequence are mentioned in this web site.
Oligo
primers for PCR are also designed in this web page (see OLIGO button).
Construction for amiRNA precursor
For constructing amiRNA precursors, fragment
I, II, and III were amplified using primers hybridizing miR319a
(miR319a-F: CACCACAAACACACGCTCGGACG [CACC at the 5' end is for pENTR/D-TOPO] and miR319a-R:
CCTATCCATGGCGATGCC)
and the 4 oligo primers designed above (miR-s~miR*a). PCR enzyme KODplus
(Toyobo) and miR319a precursor fragment plasmid cloned into pRS300 as a
template were used (Schwab et al., 2006). The amplified fragments were
agarose-gel extracted using QIAquick Gel Extraction Kit (QIAGEN) to exclude the
primers and template DNA, and were fused by PCR using the miR319a-F and
–R.
Primary-amiRNA
Cloning amiRNA precursor into estradiol
inducible vector pGX6 or pGX8
Resulted primary-amiRNA fragment was cloned
into pENTR (invitrogen), and sub-coned into pGX6 (or pGX8)-Gateway destination
vector (See chapter * in this manual). The resulted plasmid is digested to
linearize for transformation of Physco.
RT-PCR to examine knock-down of targeted
RNA by induced amiRNA
Physco transgenic lines expressing amiRNA precursor
were treated by 2uM estradiol for 2 to 9 days, for total RNA extraction and
phenotype observation. Total RNA was extracted with RNAeasy (Qiagen) from the
total tissues (including protonema, gametophore, and rizhoid) at 2 days-treated
moss. 1ug Total RNA was used for reverse transcription using Ready-To-Go You-Prime First-Strand Beads (GE healthcare). Reaction mixtures were diluted
to 5 folds before used for PCR. Primers upstream of the cleavage site were
designed for PCR. We use KODplus (Toyobo) for semi-quantitative RT-PCR, and QuantiTect SYBR Green PCR Kit (QIAGEN) and the 7500 Real Time PCR
system (Applied Biosystme) for quantitative RT-PCR (QRT-PCR). The delat-delta method was
used to calculate the relative difference among sample in QRT-PCR.