I am trying to design a morpholino to knockdown a Xenopus laevis gene. Since laevis is allotetraploid, I want to find all the possible alleles of this gene and possibly design one morpholino (or more, if necessary) that efficiently block all of the alleles. Has anyone experience in designing Xenopus laevis morpholinos? How should I look into the EST database?
Thank you!
To find X. laevis alloalleles for morpholino design and other purpose go to Xenbase (www.xenbase.org). Type the name of your gene of interest and see if it has both copies (before termed a and b now l and s). Click on each copy to see the sequence associated with the link. Blast on NCBI (nucleotide blast) it to make sure that it is indeed your gene of interest.
If both copies are not listed on Xenbase, you have several options. The first would be to blast the sequence for your gene of interest (either copy) in Xenbase and NCBI to see if you find the second copy. They will usually be the top two hits of the search and with high % similarity to each other. A good indication that they are alloalleles is that the gene copies are found in different gene scaffolds so note the scaffold number to make sure they are different. Another good indication that they are alloalleles is synteny (same genes surrounding each copy). So also check for synteny of your gene with the genes surrounding it. When you find your sequences, download and align using ClustalW or any other alignment algorithm of your preference.
If blasting does not help you find your gene of interest, then you can blast the sequence you have against the EST database on NCBI. Download all EST sequences as a FASTA file and align with your sequence. If you can't find ESTs you can go to Mayball RNA Seq assemblies (University of Texas Austin) and find the RNA transcripts containing your sequence. Follow same procedure as with ESTs.
Remember that if your MO target is in the 5'UTR, once you find your gene, you will need to download the sequence near the ATG to align both copies in this region.
If I understood correctly, you would like to design a morpholino(s) that would target a stretch of nucleotides in an mRNA of interest that would be identical between all four alleles. Since to my knowledge, sequencing of the X. laevis genome is not completed yet (http://www.marcottelab.org/index.php/Xenopus_Genome_Project), I wonder if it would be possible to find stretches of nucleotides in exons of your gene that would be identical between X. laevis and X. tropicalis? You could then design morpholinos against such highly conserved regions of nucleotides. My assumption is that high interspecies sequence conservation would imply a lower probability of allelic variability (which could still take place in any case).
To find X. laevis alloalleles for morpholino design and other purpose go to Xenbase (www.xenbase.org). Type the name of your gene of interest and see if it has both copies (before termed a and b now l and s). Click on each copy to see the sequence associated with the link. Blast on NCBI (nucleotide blast) it to make sure that it is indeed your gene of interest.
If both copies are not listed on Xenbase, you have several options. The first would be to blast the sequence for your gene of interest (either copy) in Xenbase and NCBI to see if you find the second copy. They will usually be the top two hits of the search and with high % similarity to each other. A good indication that they are alloalleles is that the gene copies are found in different gene scaffolds so note the scaffold number to make sure they are different. Another good indication that they are alloalleles is synteny (same genes surrounding each copy). So also check for synteny of your gene with the genes surrounding it. When you find your sequences, download and align using ClustalW or any other alignment algorithm of your preference.
If blasting does not help you find your gene of interest, then you can blast the sequence you have against the EST database on NCBI. Download all EST sequences as a FASTA file and align with your sequence. If you can't find ESTs you can go to Mayball RNA Seq assemblies (University of Texas Austin) and find the RNA transcripts containing your sequence. Follow same procedure as with ESTs.
Remember that if your MO target is in the 5'UTR, once you find your gene, you will need to download the sequence near the ATG to align both copies in this region.
Thank you so much for your help guys, I think I already found the alloallele :)
Thanks Enrique. The folks at the National Xenopus Resource taught me well. Gabriele I am really glad that you found your alloallele.
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