From 20/01/2014 to 14/02/2014 I’m staying at Casey Dunn’s Lab to analyze RNAseq data from priapulid development and acoel phylogenomics.
Everything I’m doing is documented on GitHub: https://github.com/nelas/bcv-at-brown
Set Azakanpaullone treatment for bilateral gastrula to see how it affects lobe development.
| CTRL FSW | 1 µM Az | 10 µM Az |
Considering my last observations of the vivo-morpholinos I’m setting up a new experiment using a lower concentration of pronase, electroporating twice (because I could not figure out how to do longer pulses), and properly washing out the pronase after 1h. Default settings:
Vivo-Morpholino: 25 µM
Pronase: 5 µg/mL
Washing after: 1h
Electroporation: 50 V, 25 µF, 1000 resistance
Actual pulses:
| CTRL FSW | CTRL Pronase | EN TB Pronase | EN SB Pronase | WNT1 TB Pronase | WNT1 SB Pronase |
| CTRL Electroporation | EN TB Electroporation | EN SB Electroporation | WNT1 TB Electroporation | WNT1 SB Electroporation |
Use 25 µM concentration for vivo-morpholinos.
| CTRL FSW | ||||
| CTRL CFSW 1h | Ttra_en_TB_vMO 25 µM | Ttra_en_SBe2i2_vMO 25 µM | Ttra_wnt1_TB_vMO 25 µM | Ttra_wnt1_SBe2i2_vMO 25 µM |
| CTRL Pronase 10 µg/mL 1h | Ttra_en_TB_vMO 25 µM | Ttra_en_SBe2i2_vMO 25 µM | Ttra_wnt1_TB_vMO 25 µM | Ttra_wnt1_SBe2i2_vMO 25 µM |
| CTRL Electroporation in CFSW | Ttra_en_TB_vMO 25 µM | Ttra_en_SBe2i2_vMO 25 µM | Ttra_wnt1_TB_vMO 25 µM | Ttra_wnt1_SBe2i2_vMO 25 µM |
BioRad Gene Pulser 50V, 25 µF, resistance set to infinity.
[photo]
| ms | |
| ctrl | 1.1 |
| en tb | 1.0 |
| en sb | 1.1 |
| wnt1 tb | 1.2 |
| wnt1 sb | 0.9 |
Higher concentration of vivo-morpholinos at higher temperatures showed no specific effect on the embryos. So it seems that the vivo-morpholinos are not penetrating the embryos. I’ll try different methods to permeabilize the live embryos to make the oligos go into the cells. List of alternatives:
| CFSW | 0.2 % Cystein | Pronase 10 µg/mL | Pronase 1 µg/mL | Sodium citrate / Sucrose | Control |
Embryos seem unaffected by calcium-free sea water, but were dead instantly in cystein. They are swimming normally in pronase treatments and stuck in the sucrose…
2h later: CFSW have abnormally spinning embryos, cystein are still dead, pronase treatments are swimming normally and look normal, sucrose have exploded. I split half volume of the CFSW and Pronase 10µg/mL and washed 3x with FSW.
CFSW: embryos are trilobed, but spinning without direction and with tissue falling off.
CFSW washed after 2h: look like regular trilobed with directional swimming. They seem to have recovered from the calcium-free experience.
Pronase 10 µg/mL: deformed embryos, somehow they failed to elongate and form lobes, even though there is tissue burst where the apical lobe would be.
Pronase 10 µg/mL washed after 2h: look like normal trilobed larvae, except that when compared to the control larvae they seem to be curved slightly and not swimming much off the bottom.
First experiment with vivo-morpholinos showed that 1-25 µM concentration between 7.0-8.5 °C resulted in no observable phenotype (morphological or behavioral) in the embryos. Thus, I’m setting up a new batch using higher temperatures and higher concentration. Female 18.
| FSW CTRL | Ttra_en_TB_vMO 50 µM | Ttra_en_SBe2i2_vMO 50 µM | Ttra_wnt1_TB_vMO 50 µM | Ttra_wnt1_SBe2i2_vMO 50 µM | |
| STD CTRL vMO 100 µM | Ttra_en_TB_vMO 100 µM | Ttra_en_SBe2i2_vMO 100 µM | Ttra_wnt1_TB_vMO 100 µM | Ttra_wnt1_SBe2i2_vMO 100 µM |
| FSW CTRL | STD CTRL vMO 25 µM | Ttra_en_TB_vMO 25 µM | Ttra_en_SBe2i2_vMO 25 µM | Ttra_wnt1_TB_vMO 2 µM | Ttra_wnt1_SBe2i2_vMO 25 µM |
| Ttra_en_TB_vMO 50 µM | Ttra_en_SBe2i2_vMO 50 µM | Ttra_wnt1_TB_vMO 50 µM | Ttra_wnt1_SBe2i2_vMO 50 µM | ||
| Ttra_en_TB_vMO 100 µM | Ttra_en_SBe2i2_vMO 100 µM | Ttra_wnt1_TB_vMO 100 µM | Ttra_wnt1_SBe2i2_vMO 100 µM |
Picked the embryos and incubated the plates.
Embryos at RT have reached trilobed stage in the morning and look normal at 25 °C. Embryos at higher concentrations 50 and 100 µM are delayed and still around bilateral/bilobed.
I fixed RT plate for PCR (RNAlater).
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.
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
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
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
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
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.
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.
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.