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  • Bruno Vellutini 03:50 on 2014/01/13 Permalink
    Tags: engrailed, , ,   

    2nd try for vivo-morpholinos in Terebratalia 

    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.

    10 °C plate

    FSW CTRL STD CTRL vMO 50 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
    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

    RT plate 18 °C

    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
    STD CTRL vMO 50 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
    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

    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).

  • Bruno Vellutini 21:00 on 2014/01/10 Permalink
    Tags: engrailed, , ,   

    Terebratalia vivo-morpholinos engrailed/wnt1 

    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


    Original sequence: Doutorado:Terebratalia transversa:Loci:Ttra.rna.tri.8524.1
    5′ UTR + (ATG) + 25 coding bp:


    Selected oligo:

    @morpholino — BLAST results

    @mismatch — BLAST results


    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:
    Selected oligo:

    @morpholino — BLAST results

    @mismatch — BLAST results

    Ttra wnt1

    Translation blocking

    Original sequence: Doutorado:Terebratalia transversa:Loci:Ttra.rna.tri.7468.1
    5′ UTR + (ATG) + 25 coding bp:


    Selected oligo:

    @morpholino — BLAST results

    @mismatch — BLAST results


    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:
    Selected oligo:

    @morpholino — BLAST results

    @mismatch — BLAST results


    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.

  • Bruno Vellutini 10:36 on 2013/12/16 Permalink
    Tags: , engrailed, ,   

    Propidium+Phallacidin and Murray Clear for Novocrania engrailed fluorescent in situ 

    Since mounting with TDE was not so good (crystals and some weird noisy background) I am staining with propidium iodide and phallacidin and mounting with murray clear. This should give a clean embryo with the mesodermal signal visible enough for details. Fluorochrome for engrailed is Cy5.



  • Bruno Vellutini 17:45 on 2013/11/23 Permalink
    Tags: engrailed, ,   

    Novocrania engrailed fluorescent in situ 

    Began a fluorescent in situ of Novocrania engrailed to better understand the mesodermic expression. The only difference this time was 10 min of half-concentrated proteinase-k (1.25 µL in 5 mL) instead of 5 min.


    Diluted new probe and added to the sample.


    Wash day went on smoothly. Added 1:100 anti-dig-pod antibody and incubated in the cold room.


    Wash off antibody.

    • Developed with TSA Cy5 for 2h.
    • Stopped reaction with 2x 5 min in detergent solution at 62 °C.
    • 1x 5 min in detergent solution at RT.
    • 3x 5 min PTw at RT.
    • Selected 6 embryos of different stages and went through TDE steps until final 97% + DAPI.
    • Letting overnight in cold room.


    Crystals have formed and are sticking around the embryos… Mounted a slide and it works, although there is background.


    Confocal at 8h!

    Nano en TDE 1.lif - Series004 s1 Nano en TDE 1.lif - Series004 s2 Nano en TDE 1.lif - Series004 s3

  • Bruno Vellutini 17:03 on 2013/10/27 Permalink
    Tags: engrailed, , ,   

    Novocrania in situ 3rd try 

    Third try with engrailed, wnt1, and wnt5. This time using half-concentrated 5 min ProteinaseK treatment. They should not get damaged!

    • Fixed for 1h25min.
    • Washed normally and incubated in pre-hybe.


    Added probes at 11h.


    Washing day embryos looking good.


    Washed out antibody with long 30 min washes.


    Started developing. Engrailed began to appear after 2hs, but nothing in the wnts. I exchanged the AP substrate every 2 hours. At 18h I transferred to new wells because crystals were forming. At 19h30 I exchanged the AP and put them in the cold room.


    Stopped engrailed and continued developing the wnts during the day. No signal came up except for background. Paused in AP without magnesium.


    Restarted the wnts to see if something comes up…


    Stopped wnts… Ethanol washes and added glycerol+DAPI overnight in cold room.


    Mount slides. Engrailed has some shitty background… Remember next time to stop before overnight.

  • Bruno Vellutini 13:20 on 2013/10/21 Permalink
    Tags: , engrailed, , , , ,   

    Terebratalia additional double in situs 

    Started Terebratalia double in situs with the following selected probes. Everything looks ok.

    wnt1 (DNP) + wnt4 (DIG) en (DNP) + wnt7 (DIG)
    wnt1 (DNP) + wnt8 (DIG) en (DNP) + pax258 (DIG)


    • Added probes to Terebratalia doubles.


    • Terebratalia doubles washing day.
    • Add Terebratalia first antibody anti-dig-pod 1:250.


    • Terebratalia doubles wash off first antibody.


    • Terebratalia doubles develop first probe and inactivate.
    • Terebratalia doubles incubate with second antibody.


    • Terebratalia doubles wash off second antibody.


    • Terebratalia doubles develop second probe.
    • Terebratalia doubles stain with phallacidin.
    • Terebratalia doubles embed in TDE+DAPI.


    • Mount Terebratalia doubles.


    • Confocal slot for Terebratalia doubles.


    Here are some scans.

  • Bruno Vellutini 09:55 on 2013/10/15 Permalink
    Tags: engrailed, , ,   

    Novocrania in situ en wnt1 wnt5 2nd try 

    Second chance on the Novocrania in situ which failed last time. This time I’ll do 7 minutes of ProtK and wash extensively with glycine to stop the reaction.

    3 embryos of each stage: radial, bilateral, early larva, and larva.

    engrailed wnt1 wnt5
    • Initially in MeOH.
    • 1x 5min 60% MeOH 40% PTw.
    • 1x 5 min 30% MeOH 70% PTw.
    • 4x 5 min PTw.
    • ProteinaseK for 7 min.
    • Stopped exactly at 7 min with 900 µL of glycine.
    • 1x 5 min glycine.
    • 1x 5 min 1% TEA.
    • 1x 5 min 1% TEA + Ac. An. –> embryos stick a bit to bottom.
    • Added 100 µL of Ac An. embryos continue to stick.
    • Washed 2x PTw 900 µL.
    • Fixed in 4% FA for 1h25.
    • Washed 3x PTw.
    • 1x 10 min PTw at 80 °C.
    • 1x 5 min PTw.
    • 1x 10 min Hybe Buffer at RT.
    • Exchanged hybe and incubate at 62 °C.


    • Added probes.
    • Embryos were still looking ok, although somewhat transparent.


    • Larvae look damaged and may fall apart. Saw a bilateral missing a piece… Otherwise it looks better than last time. I think the time or concentration of ProtK (newly opened) really needs to be decreased!
    • 10 min and 40 min washes with hybe wash.
    • Series of 30 min hybe to 2x SSC.
    • 3x 20 min 0.2X SSC + 0.1% Tween-20.
    • Grade-series of 0.2X SSC to PTw 10 min each.
    • 5x 5 min PBT.
    • Incubate 1h with 1x blocking buffer made with the last 500 µL of 5x blocking buffer from the fridge and newly made maleic acid buffer.
    • Exchanged for 1:5000 Anti-DIG-AP in blocking buffer and incubated overnight in cold room.


    Embryos still look integer although larvae are damaged.

    • Started washing with PBT at 13h.


    • Began to develop at 16h.
    • Faint band of engrailed at 19h.
    • Noticed some precipitated when exchanging NBT/BCIP, maybe old BCIP (it is running out)?
    • Let overnight in the cold room.


    • Checked at 11h and the engrailed stripe was more prominent; nothing yet in the others.
    • There are crystals forming in the bottom :( and some clear precipitate floating around…
    • I exchanged the AP substrate and let it go under RT.
    • Exchanged again after 2h.
    • Final exchange and cold room at 17h30. Maybe there was a bit of background coming up in engrailed… not sure so I let it overnight and will stop tomorrow.


    • engrailed well began to show some background and the others were looking bad with crystals packing up. So I stopped them all.
    • AP -magnesium 2x.
    • Ethanol washes.
    • 70% Glycerol exchanged once for glycerol with 1:5000 DAPI.
    • I restarted the wnts because I felt bad. Let them develop overnight.


    • Stopped wnts and did alcohol series.
    • Mounted and took photos of engrailed.
  • Bruno Vellutini 18:36 on 2013/09/24 Permalink
    Tags: engrailed, , , ,   

    Novocrania in situ en, wnt1, wnt5 

    Started an in situ with Novocrania! I picked 3 specimens of each stage making 12 embryos in total. The stages are: radial, bilateral gastrula, early larva and larva.

    Nano en Nano wnt1 Nano wnt5

    All 12 embryos per well survived the first day.


    Since engrailed probe had low concentration I diluted what I had (13 ng/µL) into 200 µL of hybe (resulting in 0.5 ng/µL) and added anyway. I’m synthesizing more so that I can add tomorrow.


    Changed engrailed hybe with nice new probe. However, embryos are really transparent. Larval morphology also does not look normal. I think something is wrong.


    Engrailed well has not any normal looking embryos anymore. They are all falling apart. In the other 2 wells there are still 4-5 embryos that look ok, but fragile.

    I continued with the washes anyway and incubated with the antibody overnight at 4 °C.

  • Bruno Vellutini 14:52 on 2013/09/22 Permalink
    Tags: engrailed, , ,   

    Probe synthesis of Novocrania segmentation 

    After successfully cloning Novocrania genes using Andi’s primers it is time for probe synthesis.

    Set Probe PCR for en, wnt1 and wnt5.


    Gel and extraction went fine, except that I forgot the dna ladder…

    2013-09-23 23.37.45

    Extracted the probe PCR.


    Began probe synthesis transcription reaction at 10h and stopped at 16h. Everything normal.


    After centrifuging there was a very small pellet for engrailed. Wnt1 and wnt5 had normal pellets. Here are the nanodrop:

    Plasmid Gene Enzyme ng/µL
    BV267 engrailed SP6 13.08
    BV271 wnt1 SP6 156.92
    BV274 wnt5 SP6 54.99

    Since engrailed was so low I started a new transcription reaction at 12h.


    Good engrailed concentration! 1334 ng/µL.

  • Bruno Vellutini 01:01 on 2013/09/10 Permalink
    Tags: engrailed, , ,   

    Novocrania one more time 

    Using Andi’s primers with annealing temperature at 57 °C.


    Engrailed rendered a band at the expected size (638 bp), although it is a bit spread band. It is quite possible that wnt1 had a band at the right place (718 bp), but it was very weak; I cut it anyway. Wnt5 band was also at the correct place (932 bp), but also weak.

    2013-09-11 16.37.23

    Set ligation reaction at 16h and stored at 4°C.


    Transformation and plating.


    en had a lot of colonies, wnt1 had a normal amount, and wnt5 had a few colonies. I managed to do 10 colonies for the first two and 8 colonies for wnt5:

    2013-09-16 10.57.37


    Picked colonies for minipreps

    gene colony T7
    en 8 BV266
    en 9 BV267
    en 10 BV268
    wnt1 1 BV269
    wnt1 2 BV270
    wnt1 3 BV271
    wnt5 1 BV272
    wnt5 2 BV273
    wnt5 3 BV274


    • Extracted plamids.


    • Set sequencing pcr at 10:30.
    • Dropped sequences at the facility at 14:30.


    Checked the alignment and it looks good! See files: Nano_en-wnt1-wnt5

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