Setting up the first vivo-morpholino assay in Brachiopoda. Here is a description of the oligos prepared by Gene Tools for engrailed and wnt1 genes in Terebratalia.
Ttra engrailed
Translation-blocking
Original sequence: Doutorado:Terebratalia transversa:Loci:Ttra.rna.tri.8524.1
5′ UTR + (ATG) + 25 coding bp:
GACAAGTTTTAATTAATATATGGTTGGATATCGGGACATGCTCTGCGTTGTAATCCACTG
ATCAGCTGTTAGCCGGCCTTTGATACAGATACAGGGACTATGCGAATGTCATTTGGAGGA
ACTAACCACGTCTGGACAACAGTATTATCAGTGAACCTCGGAAAACAGTGAGACGATTCT
CAGAGACAATTTCTATAAACTATACTGAACTAGTTAACATTTTATTTAACAATTTTCCAA
AGGCTGACCCGATATTGATATATCCGGATATACAGGAGAAAACATATCTATAAACAGTCT
GTACT(ATG)ACACTCATACGAATGGATGTGAATG
Selected oligo:
GGATATACAGGAGAAAACATATCTATAA[ACAGTCTGTACT(ATG)ACACTCATAC]GAATGGATGTGA
@morpholino — BLAST results
GTATGAGTGTCATAGTACAGACTGT
@mismatch — BLAST results
GTtTcAGTcTCATAcTACAcACTGT
Splice-blocking
Genomic Ttra en consensus alignment: ~/Biologia/Doutorado/Terebratalia/molecular/morpholinos/Ttra_en_genomic_consensus.aln
25 bp exon + 25 bp intron (position: 17291-17340) — Ttra_en_mo_e2i2:
CAAGATATTCTGACCGACCGTCGTCaggtaaggaattataataatacgtt
Selected oligo:
CAAGATATTCTGACCG[ACCGTCGTCaggtaaggaattataa]taatacgtt
@morpholino — BLAST results
TTATAATTCCTTACCTGACGACGGT
@mismatch — BLAST results
TTAaAAaTCgTTACCTcACcACGGT
Ttra wnt1
Translation blocking
Original sequence: Doutorado:Terebratalia transversa:Loci:Ttra.rna.tri.7468.1
5′ UTR + (ATG) + 25 coding bp:
GGCAGATCCTCAGATCGTATCTTGTCCCTGTTTATAGTCAGTCATGTTCATGTAGAACAC
GTATGTTGTGATTCCCTTTTCTTGTGTTGCACGGATGTACGACAGCTGTATTTGAAAAGC
GACAGCCCGATTGATGAACACTGGGGAACAATGCCGACGCGACATCAAAAGAACTTTTTA
TCAACACGAGGTATTTTAACGTTAATACAGTAGCAGCCTGCCTGAACGAGACCTTCGAAG
CACGAGACATTACTCTAAAATCCAAAACGGGGCGGTCTGATCCTCGATTGCTGTACAATT
TATGGTATGTCACACAATAAACTCGCACAATACACGAGTGTGATTGGCATACAAGAGCGG
CACACATCAAAAGAAGAGAAAGTTCCGCATCAAGCCTTAACATTTTACACGGATACATCG
GACAGTGAACATGTTCTTTTTATTTCGTAAGAGAAAACAGAAGCTGTCCACGTGCAGGAG
TATCAAACGTCACTAATTAATTAAAGTGCGATCTTAATTAATGTGATAAAATTTATTTTA
ACGAAACCACGTGTGGAGTAGACGATCAACCTTTTTAATTGGATTATCAGTTTTGAGCAC
CCTCTTTATTAAAAGTTTACAAAAAGATTTAAAACAGGGACTCAACACTCAGAAGTAAAC
AACATATATATACACTTACGCACACTATTGTGTTTTAAGAATACTTTATTTTTGAAATCG
TTAATCTTTGTCTGTGTATTGGGTCGCGTTACGACATTATTTACTAGTGTTGTTGTGT(A
TG)AGACAAGAATACGACATGAACATCA
Selected oligo:
TAATCTTTGTCTGTGTATTGGGTCGCGTTACGACATTATTTACTAGTGTTGTT[GTGT(ATG)AGACAAGAATACGACATG]AACA
@morpholino — BLAST results
CATGTCGTATTCTTGTCTCATACAC
@mismatch — BLAST results
CATcTCcTATTgTTcTCTgATACAC
Splice-blocking
Genomic Ttra wnt1 consensus alignment: ~/Biologia/Doutorado/Terebratalia/molecular/morpholinos/Ttra_wnt1_genomic_consensus.aln
25 bp exon + 25 bp intron (position: 5186-5235) — Ttra_wnt1_mo_e2i2:
CATTTATACAGAGGCGTACGGTGGTggtaagtaagacaataccttgtatc
Selected oligo:
CATTTATACAGAGGCGTAC[GGTGGTggtaagtaagacaatacct]tgtatc
@morpholino — BLAST results
AGGTATTGTCTTACTTACCACCACC
@mismatch — BLAST results
AGcTATTcTCTTAgTTACgAgCACC
Experiment
24-well plate (500 µL) with humid chamber and keep on incubator. vMO stock concentration: 0.5 mM. Add vMO at swimming blastula stage to prevent any initial translation. I prepared 250 µL of vivo-morpholino dilutions and added 250 µL of embryos.
FSW CTRL |
STD CTRL vMO 1 µM |
Ttra_en_TB_vMO 1 µM |
Ttra_en_SBe2i2_vMO 1 µM |
Ttra_wnt1_TB_vMO 1 µM |
Ttra_wnt1_SBe2i2_vMO 1 µM |
|
STD CTRL vMO 5 µM |
Ttra_en_TB_vMO 5 µM |
Ttra_en_SBe2i2_vMO 5 µM |
Ttra_wnt1_TB_vMO 5 µM |
Ttra_wnt1_SBe2i2_vMO 5 µM |
|
STD CTRL vMO 10 µM |
Ttra_en_TB_vMO 10 µM |
Ttra_en_SBe2i2_vMO 10 µM |
Ttra_wnt1_TB_vMO 10 µM |
Ttra_wnt1_SBe2i2_vMO 10 µM |
|
STD CTRL vMO 25 µM |
Ttra_en_TB_vMO 25 µM |
Ttra_en_SBe2i2_vMO 25 µM |
Ttra_wnt1_TB_vMO 25 µM |
Ttra_wnt1_SBe2i2_vMO 25 µM |
Incubated 20h late blastula at 7.5 °C in turned off outdoor incubator. ~2 am temperature was 8.5 °C.
11/01/2014
Checked samples during the morning. All wells had actively swimming radial gastrula and some dead embryos. FSW control and 1 µM samples had more active embryos. Letting it go longer.
18:00 All wells with swimming asymmetric gastrula. Ectoderm of larvae at 25 µM seems not perfectly integer. I observed some round flattened gastrula which did not become bilateral on the wnt1 splice-blocking 25 µM well, but might be just a coincidence.
12/01/2014
Embryos still look ok and have reached the bilateral stage. Temperature dropped to 7.0 °C.
I will fix them in RNAlater tonight or tomorrow morning.