Alternative Transformation Methods

Arabidopsis thaliana

typisch model voor plant research

small genome (135 Mb)

grootte genome A. thaliana

short life cycle (~6 weeks)

lengte life cycle A. thaliana

self-pollination

pollination bij A. thaliana

Easy genetic transformation

Simple growth in small space

Hoofdvoordelen A. thaliana

In planta transformation method, dipping flowers of the plant in an Agrobacterium suspension

floral dip

During development, the gynoecium is an ‘open vase’

In this open vase, Agrobacterium can enter and infect the ovules (female gametophyte), leading to transformed seeds

waarom werkt Floral dip bij Arabidopsis?

visual selection marker to select transgene seeds after floral dip

FAST

Oleosin protein (OLE1) accumulates in oil bodies and is fused to GFP.

principe FAST

Can be increased by targeting flowers at the right stage

hoe kan je de lage floral dip efficientie verhogen?

Gene gun/biolistics

Electroporation (protoplasts)

poly-ethylene glycol (PEG) (protoplasts)

Nanobiotechnology

voorbeelden methoden direct gene transfer

Build up a high pressure in a chamber

A rupture disk breaks at a certain pressure

A holder with DNA coated on small (gold) particles is accelerated

These particles are shot into the tissue (cells), where the DNA can enter the cells

werking gene gun

No genotype dependency for delivery (still required for regeneration)

voordelen gene gun

DNA fragmentation

Multi-copy inserts

Not very scalable

→ Very rarely used

nadelen gene gun

Gold (Au) - particles actually quite large (1 µm)

Most cells don’t survive

probleem met goud voor gene gun

Nanobiolistics: so going to smaller particles (like MSN and IONP)

oplossing probleem grootte gouddeeltjes

DNA nuclease

functie Cas9

single guide RNA with 20 bp, associates with Cas9, and is complementary with target sequence

functie sgRNA

belangrijk motif dat gevonden moet worden door Cas9

functie PAM motif

3 nucleotides upstream of PAM

waar maakt cas9 een cut in het DNA tov PAM?

The repair systems try to repair the break, but if repaired perfectly, Cas9 can cut again until a mutation (indel) occurs that prevents sgRNA binding

NHEJ can lead to insertions/deletions (indel) > mutation

link repair systems met CRISPR/Cas9

non-homologous end-joining

Most prevalent pathway in plant cells

NHEJ

Homology-directed repair (template-based!)

HDR

oligo insertion

RNP delivery

Transformation of organels (chloroplasts, protoplasts,…)

waarvoor gene gun momenteel gebruikt wordt

CRISPR/Cas9 makes DSB

Short oligonucleotides introduced in cel together with DNA encoding CRISPR/Cas9

Adding of cis-regulatory elements in promoters

oligo insertion met gene gun

ribonucleoprotein complex (Cas9 + sgRNA)

RNP

proteolistics

if gene gun is used for protein delivery

Cells contain many chloroplasts (10-100)

Chloroplasts contain multiple genomes (plastomes) (10-100) (might be mix of (non-)modified genomes (heteroplasmy))

Selection via the antibiotic spectinomycin towards homoplasmy

probleem homoplasmy / heteroplasmy bij transformation of chloroplasts

Homologous recombination is possible (prokaryotic genome-like environment)

High transgene expression (a lot of copies, no silencing [no TGS no PTGS], ...)

Maternal inheritance (no spreading of transgenes via pollen)

advantages transformation of chloroplasts

Difficult

Not for genes that have a function outside of the chloroplast

nadelen transformation of chloroplasts

Protoplasts: plant cells without a cell wall

Cell wall is removed using enzymes (e.g. cellulase), leading to a spherical shape

Only the cell membrane remains, making it easier to deliver DNA, RNA or proteins

principe protoplasts

Polyethylene glycol (PEG)

Electroporation

methodes van transfection (introduction of DNA) into protoplasts

Regeneration to plants very difficult

Very empirical

Limited to certain plant families

genomic rearrangements

moeilijkheden transformation of protoplasts

Subcellular localization

Protein production

voor wat is transient expression voldoende?

Nicotiana (tobacco)

waar wordt transient expression veel gedaan?

leaf infiltration

typisch voorbeeld van testen van transient expression in tobacco (Nicotiana)

Compact promoter:

--35 box and -10 box binding RNA polymerase

-Binding via σ⁷⁰ subunit

-Repressor proteins binds operators

hoe wordt transcriptie geregeld bij prokaryoten?

Shine-Dalgarno sequence

important sequence in translation in prokaryotes

Base pairs with 16S rRNA to start translation

Ribosome can thus start in the middle of an mRNA

polycistronic mRNA (poly, a lot; cistron, prokaryotic gene)

functie Shine-Dalgarno sequence

Recruitment of chromatin-remodeling complexes

Hoe kunnen transcription factors zorgen voor repressie?

Recruitment of polymerase II

hoe kunnen transcription factors zorgen voor activatie?

Genetic code is universal: bacterial DNA in plants will encode the same protein, but in practice expression is more complex

Codon usage differs between species → differences in tRNA abundance

This affects translation efficiency, since some codons have fewer corresponding tRNAs

waarom codon optimization?

adapt the gene to use codons that are more abundant in the host species

codon optimization

selection marker placed here, since T-DNA can be truncated at this border → selecting for complete T-DNAs

Truncated: transfer of T-DNA happens from RB to LB. Sometimes transfer stops too early and does not reach LB, resulting in incomplete T-DNA in the plant. If the selection marker is placed near RB, it can still be integrated while the rest of the T-DNA is missing. This risk is avoided when placing it near LB

waarom selection marker plaatsen bij LB?

Cauliflower mosaic virus (CaMV).

van wat komt de 35S promotor?

Strong, ubiquitously expressed

voordeel 35S promotor?

associated with gene silencing, can also affect neighboring genes

nadeel 35s promotor?

express a gene where and when it normally is not expressed

Ectopic expression

35S promoter

nos-promoter

twee exogenous promoters

bacterial origin, but from A. tumefaciens T-DNA, so adapted to be expressed in plant cells

origin nos-promotor

always active, from the plant itself (endogenous)

constitutive promoters

UBIQUITIN promotors

voorbeeld constitutive promotors

pOp6/LhGR-system (a chimeric system)

voorbeeld inducible promoters

A mutant DNA binding domain from E. coli lacI (Y17H)

GAL4 activation domain from S. cerevisiae

Ligand-binding domain of the rat glucocorticoid receptor (GR)

waaruit bestaat LhGR?

Six copies (6x) of the lac operator (lacOp)

A minimal cauliflower mosaic virus (CaMV) 35S promoter (- 50 to + 8)

waaruit bestaat pOp6?

Terminator is very important for proper mRNA processing and gene expression

belang terminator

The terminator is not always well defined, so often the whole 3’ region and some downstream sequence are used as terminator sequence

Hoe wordt de terminator sequentie gedefinieerd?

Poly(A) signal (AAUAAA) is recognized → cleavage of the RNA → poly-A tail added, downstream RNA is degraded

hoe werkt de terminator?

Weak terminators are associated with gene silencing

Wat met weak terminators?

Inhibits polypeptide synthesis (ribosomes)

werking kanamycin

E. coli neomycin phosphotransferase II (nptII) inactivates kanamycin by phosphorylation

resistentiegen tegen kanamycin

Concerns surrounding horizontal gene transfer of antibiotic resistance genes

zorgen rond selectiemarker tegen kanamycin

Binds to the 30S subunit of bacterial ribosomes and stops protein synthesis

werking spectinomycin

Resistance by: aadA or Streptomyces flavopersicus spcN

resistentiegen tegen spectinomycin

Increasingly used in organogenesis-based methods

waar wordt spectinomycin vooral gebruikt?

Used in chloroplast transformation (see earlier)

OR equipped with N-terminal chloroplast signaling peptide

wanneer werkt spectinomycin?

Glyphosate inhibits EPSPS and blocks the biosynthesis of aromatic amino acids

With an Agrobacterium CP4 EPSPS gene, plants become tolerant to glyphosate

werking glycphosate en resistentie ertegen

Glyphosate

Glufosinate

Imidazolinone-herbicides

herbicides die we gezien hebben

phosphinothricin (PPT)

ander woord voor glufosinate

PAT gene (PPT acetyltransferase)

BAR gene

twee mogelijke resistentie genen tegen glufosinate

Inhibits glutamine synthesis (via inhibition of glutamine synthetase)

hoe werkt glufosinate

Affect the synthesis of leucine, valine and isoleucine by inhibiting acetolactate synthase (ALS)

werking Imidazolinone-herbicides

endogenous mutant ALS (acetolactate synthase) allele

E.g. “HRA”: highly resistant ALS

resistentie tegen imidazolinone

Phosphomannose-isomerase

PMI

E. coli PMI converts mannose-6-P in medium to fructose-6-P

functie PMI

Accumulation of mannose-6-P is toxic to plants (so when no PMI present)

PMI als selectiemarker

Golden rice

In welke context wordt PMI vaak als selectiemarker gebruikt?

betalain

rood pigment bij RUBY reporter

tyrosine

vanwaar wordt betalain gemaakt?

2A: ribosomal skipping sequence → causes “skipping” during translation, resulting in separate proteins from one transcript

One promoter drives expression of 3 enzymes → 3 separate proteins are produced

These enzymes together convert tyrosine into betalain (red pigment) → RUBY reporter phenotype (red colour)

Werking RUBY reporter

Presence of a (herbicide) selection marker in a GMO does not mean it can be used as a trait

Often left over from making the GMO

is een selective marker ook een trait?

Cre/LoxP system

2 T-DNAs

manieren om selectie marker te removen

Cre is a site-specific recombinase, and enzyme that catalyzes DNA recombination

Cre

Drie zaken die kunnen gebeuren op LoxP sites, door Cre

Cre is expressed under an inducible promoter (e.g. HSP). Upon induction, Cre mediates recombination between LoxP sites, removing the selection marker.

auto-excision in context van Cre/LoxP system om selectiemarker te verwijderen

LoxP sites. These are placed around the selection marker

hoe ‘mark’ je een selectiemarker om te verwijderen via het Cre / LoxP system?

One T-DNA contains the GOI and another contains the selection marker

They integrate at different (unlinked) loci in the genome

Through genetic segregation (crossing), the selection marker can be separated from the GOI, resulting in marker-free plants

hoe gebruik je twee T-DNAs om een selectiemarker te verwijderen?

using two separate Agrobacterium strains (each with one T-DNA) is the easiest approach

makkelijkste manier om 2 T-DNAs te gebruiken om selectiemarker te verwijderen

Transgenesis: foreign species DNA

Cisgenesis: DNA already available to the breeder

Intragenesis: DNA already available to the breeder, but genes can be chimeric: promoter and/or terminator of other gene of that species

Cis-, trans- and intragenesis

Morphogenic regulators are genes / proteins that direct the development and regeneration of plant tissues

morphogenic regulators

Use of transcription factors BABY BOOM (BBM) & WUSCHEL (WUS)

transcriptiefactoren die regeneration kunnen promoten door somatische embryo formation te induceren

involved in embryogenesis

functie BABY BOOM

BABY BOOM is normally very specifically expressed, but here a constitutive promoter (35S) is used, causing expression in tissues where it is normally not expressed → leads to spontaneous formation of somatic embryos

hoe wordt BABY BOOM gebruikt voor de formatie van somatic embryos?

BBM and WUS are used temporarily, but need to be removed to avoid abnormal growth (pleiotropic effects)

They stimulate direct somatic embryogenesis and increase transformation frequency

Breakthrough: makes regeneration less genotype-dependent

use of BBM / WUS for maize transformation

Instead of using constitutive promoters, developmental-specific promoters (e.g. pLTP) are used to drive BBM/WUS expression → reduces pleiotropic effects

functie pLTP in context van use of BBM / WUS for maize transformation