Designer 3 is an online app for the automated design of these
artificial microRNAs.
1. Go to http://wmd3.weigelworld.org/. In the upper part, click
on the “Designer” button. Make sure that the correct genome
is selected (Araport11).
2. In the target gene box, write the Gene ID of your target gene
(e.g., “AT3G24650.1”). Enter an email address to have your
results sent to, and click “submit.” An email will be sent with a
link to your result page.
3. The results show a summary of your job at the top, followed by
a list of amiRNA sequences. The list contains the amiRNA
sequence, the hybridization energy of the miRNA to a perfectly
matching complement, the identifier of the target gene, and
the hybridization energy of the miRNA to the target site in the
target gene. These amiRNAs are ranked based on suitability
and specificity, and the best ones in green.
4. Once selected, the amiRNA sequence needs to be engineered
into an endogenous microRNA precursor for expression. The
"Oligo" tool in the webpage helps design primers for modifying the A. thaliana MIR319a precursor. Click on the “Oligo”
button, paste your selected miRNA sequence, select the RS300
vector, and click the “submit” button. This will provide the
oligo sequences necessary to proceed with the cloning. Further
information about the cloning strategy is located in the
“Download” section of the webpage.
3.12.3 SIGnAL T-DNA
Express
Perhaps one of the most powerful resources available to Arabidopsis geneticists are T-DNA mutant collections. These collections
were created by large-scale Agrobacterium tumefaciens transferDNA (T-DNA)-induced insertional mutagenesis during the late
1990s and early 2000s [reviewed in 81]. Because T-DNA inserts
contain known sequences, primer design for genotyping assays is
relatively straightforward and can allow for direct mapping to the
genome to precisely identify the insertion locus. In the following
steps, we will describe how to identify a SALK line mutant for ABI3
and design its genotyping assay. While we primarily focus on the
SALK collection, there are also the GABI-KAT, SAIL, and WISC
lines available for T-DNA mutants. All of the mutant collections are
in the Col-0 background. For a more detailed description of the
T-DNA collections [82]:
1. Go to http://signal.salk.edu/cgi-bin/tdnaexpress. You will see
a genome browser showing gene models as green lines. Below
the gene models, you will see graphical representations of genes
and insertion mutants associated with that particular genomic
region. Your goal here is to find T-DNA mutants for your gene
of interest, ABI3. Under “1. Search,” enter “AT3G24650” as
your query.
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G. Alex Mason et al.
artificial microRNAs.
1. Go to http://wmd3.weigelworld.org/. In the upper part, click
on the “Designer” button. Make sure that the correct genome
is selected (Araport11).
2. In the target gene box, write the Gene ID of your target gene
(e.g., “AT3G24650.1”). Enter an email address to have your
results sent to, and click “submit.” An email will be sent with a
link to your result page.
3. The results show a summary of your job at the top, followed by
a list of amiRNA sequences. The list contains the amiRNA
sequence, the hybridization energy of the miRNA to a perfectly
matching complement, the identifier of the target gene, and
the hybridization energy of the miRNA to the target site in the
target gene. These amiRNAs are ranked based on suitability
and specificity, and the best ones in green.
4. Once selected, the amiRNA sequence needs to be engineered
into an endogenous microRNA precursor for expression. The
"Oligo" tool in the webpage helps design primers for modifying the A. thaliana MIR319a precursor. Click on the “Oligo”
button, paste your selected miRNA sequence, select the RS300
vector, and click the “submit” button. This will provide the
oligo sequences necessary to proceed with the cloning. Further
information about the cloning strategy is located in the
“Download” section of the webpage.
3.12.3 SIGnAL T-DNA
Express
Perhaps one of the most powerful resources available to Arabidopsis geneticists are T-DNA mutant collections. These collections
were created by large-scale Agrobacterium tumefaciens transferDNA (T-DNA)-induced insertional mutagenesis during the late
1990s and early 2000s [reviewed in 81]. Because T-DNA inserts
contain known sequences, primer design for genotyping assays is
relatively straightforward and can allow for direct mapping to the
genome to precisely identify the insertion locus. In the following
steps, we will describe how to identify a SALK line mutant for ABI3
and design its genotyping assay. While we primarily focus on the
SALK collection, there are also the GABI-KAT, SAIL, and WISC
lines available for T-DNA mutants. All of the mutant collections are
in the Col-0 background. For a more detailed description of the
T-DNA collections [82]:
1. Go to http://signal.salk.edu/cgi-bin/tdnaexpress. You will see
a genome browser showing gene models as green lines. Below
the gene models, you will see graphical representations of genes
and insertion mutants associated with that particular genomic
region. Your goal here is to find T-DNA mutants for your gene
of interest, ABI3. Under “1. Search,” enter “AT3G24650” as
your query.
80
G. Alex Mason et al.