Isolation of rice gametes …and other stuff

Lab meeting, 11-02-16Li, Chenxin

Intro/Review (Gametes)

• The male and female gametes are very different in terms of size and gene expression.

• Egg and sperm express different histone variants and histone modifiers. Difference in chromatin landscape has to be reconciled after fertilization.

• (Anderson et al. 2013)

Intro/Review (Isogenic Zygotes)• Zygote inherited a lot of transcripts from the egg.

Egg and zygote cluster together (red circle).

Anderson et al. 2016, unpublished

Intro/Review (Isogenic Zygotes)

• Minor wave of gene expression at 2.5hap (by karyogamy), major wave of gene expression at 5hap (nucleoli fusion).

• The zygotic genome activates early. (ZGA happens early in plants, whereas in animal ZGA can take a few cell division.)

Anderson et al. 2016, unpublished

Intro/Review (Hybrid Zygotes)

• Hybrid zygotes from indica and japonica subspecies (~1% SNP), both directions (JxI and IxJ).

• Most genes maternally biased in both direction of cross (red circle).

• A few genes paternally biased (blue circle).

• Paternal expressed genes may play important roles in embryogenesis (WOX8/9, BBMs).

Anderson et al. 2016, unpublished

How was the parental expression pattern established (Hypothesis)?• Most genes maternally biased and a few genes paternally biased.

Activation marks

Repression marks

Egg: genome wide activation marks, local repression marks

Sperm: genome wide repression marks, local activation marks.

Zygote: transiently different chromatin, resolved during seed development.

Genome wide♀ biased

locally♂ biased

This model can be tested by small RNA seq of gametes and zygotes, also by Bisulfite seq(methylome). Can’t do ChIP-seq, because can’t get enough to precipitate.

Project workflow

Collect cells

Make Library

Look at Data

1) Egg isolation2) Sperm isolation3) Zygote

Everything is tricky, and it’s all about tricks.

Sequence the heck out of it

Isolation of Egg

• 3 or more replicates, each 30+ cells.

Collect flowers

Dissect out carpels

Push out egg

Isolation of Egg – Collect Flowers

• Which flowers to get? -Get the flowers that are going to open today.

1

2

34

5

6

78

9

Rice flowers at 10:30am or so. Go to the greenhouse early. Based on the position on the panicle, I can tell if the flower is going to open today or not.

Collect appropriate flowers into a tube of 0.3M mannitol.

Isolation of Egg – Collect Carpels

Photo credit: Zach

A rice flower has two bracts: the larger is called a lemma; the smaller is called a palea.

1) Remove lemma, the carpel will always stick to palea;

2) Remove stamens;3) Tear apart the palea along

its symmetric axis, which gives me access to the base of carpel;

4) Gently detach the carpel at its base.

Isolation of Egg – Collect Carpels

Video credit: Zach

I have get used to the size of the carpel; so I don’t have to use a dissection scope;

It’s very important not to deform or crush the carpel! –Be very gentle.

Isolation of Egg – Collect Carpels

The carpels sit in a boat of 0.3M mannitol until they are further dissected.

How to get the egg out?

Sy

CC

Sy

EC

Carpel

CutEC

Ovule

Push Egg – Cutting the Carpel Getting to the really tricky part.

• Dissection made in a 6ul droplet of 0.3M mannitol, the droplet has to bead up.

Rinse the slide in DI water while rubbing the slide very hard with my thumb.

Push Egg – Cutting the Carpel

• Amount the carpel into the droplet, and cut through it.

Dissection scope: eyepiece 10X & scope 3.5X.

Picture taken with my iPhone 4s.

It has to be a clean and sharp cut. Don’t crush or deform the carpel.

Push Egg – Cutting the Carpel – the Razor

Double side blade. Divide each side into 5 spots: 1 – 5 and 6 – 10. Each spot is used only once.

Push Egg – Inverted Microscope

Cut in this droplet

Move the basal part of the cut carpel to a new droplet

Push Egg

Picture taken with my iPhone 4s, eyepiece 10X, object lens 5X.

Use an acupuncture needle to gently push the carpel to loosen up the inside of the ovule.

What does the Egg Look Like?

Anderson et al. 2013

Uchiumi et al. 2006

The egg cell is asymmetrical, with nucleus at one side of the cell and vacuole at the opposite side, ~50uM diameter.

Picture taken with by phone, eyepiece 10X, object lens 40X.

Pictures taken with by phone, eyepiece 10X, object lens 40X.

Pictures taken with by phone, eyepiece 10X, object lens 40X.

Pick up the Egg

Fine capillary connected to a transfer pipette. Aim at the egg cell and pick it up. (But DON’T touch the egg or slide!)

Transfer to a tube. Snap Freeze in liquid N2. The tube (the sample) is always kept in dry ice (or -80).

Egg Isolation Summary

• Things are fragile; everything has to be very careful and gentle; every step is important;

• About 8-10 egg cells from 30 unopen flowers a day.• I need 3+ replicates, each 30+ egg cells. • I completed the first and second replicates (each 35 cells), started the

3rd replicate. • Will make 4 replicates total. • If things go well, will finish by the end of this quarter.

Sperm Isolation

• Sperm isolation happened in University of Oklahoma, Scott Russell’s lab.

• Protocol was developed by Xiaopin Gou (Gou et al. 1999).• Protocol takes 15-16 hours, and has to be done in a day. One day per

replicate.

Sperm IsolationCollect 60 or so panicles into a water bucket. 7am.

Dissect out the anthers into a tube (>3mL). 7am – 4pm

Open anthers in cold 45% sucrose by pressing the anthers against the tube using a glass rod.

100um filter (remove anther)

Burst pollen in cold 15% sucrose 20min.

30um filter (remove pollen)

(15% Percoll +15% sucrose)/ (40% Percoll + 15% sucrose) gradient (separate vegetative cells) 4800g 1hr

Spin down and rinse pellet though 10um filter in 15% sucrose.

30um filtrate15%P + 15%S

40%P + 15%S

30um filtrate

Sperm Isolation cont.

Spin down in 800ul 15% sucrose (remove Percoll)

Freeze in -80. 10pm.

A successful prep should have ~5cells/ul, and hundreds of cells in total.

Having >3ml anthers to start with is very important, because we need to see a visible band or pellet after the centrifuge steps. The gradients only work with glass tubes, not plastic. All the solutions have to be kept cold during the protocol.

Layer on (40% Percoll + 15% sucrose) gradient (remove organelles)

Take 75ul – 100ul from the bottom.

40%P + 15%S

Take 2ul to count under microscope

10um filtrate

Project workflow

Collect cells

Make Library

Look at Data

1) Egg isolation2) Sperm isolation

1) RNA extraction2) Library

construction

Protoplasts as “mock” materials

• I want to practice the protocol before I use my real materials. (After we figure out which kits we’re going to buy.)

• Both egg and sperm are protoplast-like: weak/no cell wall, do not allow turgor.

• Protoplasts are scale-able: I can count cells and dilute them. • For RNA-extraction from protoplasts, no grinding is needed, just like

from egg or sperm. • Protoplasts can be made from Arabidopsis leaves or rice seedlings.

Protoplast. It’s free.

• We have a bottle of cellulase powder in the 4-degree from years ago, and it’s still good.

• I borrowed Macrozyme from Harada Lab. (0.075g for 30ml)• A few years ago a company gave us a sample of 100um filter, which I’m

going to reuse forever.

RT, 60rpm 2hr

Take a sample and look at it under microscopeProtocol from Harada Lab

Cut 60 Arabidopsis leaves

Picture taken by my phone, eyepiece 10X, object lens 40X.

Protoplast

Rinse through 100um filter. The filter is now kept in 20% EtOH, and will be reused. I wash it with DI water before and after each use.

200g, 2min

Remove supernatant and count cells, aliquot and freeze.

Protoplast – Quantification

Stick a reinforcement label on a slide

Divide the circle into quadrants; load 2ul of 1/10 dilution; count.

~200 cells in 2ul (1/10); 1000/ul in original (3ml) total. Aliquot 20000, 2000, 200 and 20 and freeze. Each in triplicates. I may also try to make protoplasts from rice seedlings.

DRM2 CRISPR

• OsDRM2 (Os03g02010), the CHH (de novo) cytosine transferase in rice; gets commands from small RNA (Moritoh et al. 2012).

• CDS is 1524bp, which is 507aa. • Two gRNA designed at the first exon, nucleotide position 81 and 130.

I have two transformants back. One of them seem to be wildtype. Having some problem with TOPO

Acknowledgment

• Sundar Lab• -Special thanks for Bao in helping me on greenhouse work. • Russell Lab at University of Oklahoma