Transgenosis
methods of transformation
Foreign DNA introduction
• transient
DNA is / is not
integrated to chromosom/plastom/chondriom)
• stable
Stable integration results in:
• mutation (always, but negative effects are rare)
• expression of introduced gene (if intended, allways
unsure – RNAi, …)
Expression of introduced gene
- universality of genetic code allows functional protein
synthesis even from genes of distant organisms
- specific regulatory sequences necessary for transcription
and translation
PROMOTER:
- start site and direction of transcription
- interacts with trans elements (transcription factors = specific spatiotemporal regulation of activity – specific host promoters)
- special promoters: constitutive, inducible (EtOH, heat, estradiol)
- origin: plants, plant pathogenes, synthetic
TERMINATOR - balanced with promoter!
Translation
AACAATG
Stop
mRNA
coding sequence
-
codon usage can differ (tRNA
frequency)
- problematic if expressing plant
gene in bacteria
-
ATG sequence context –
important!!!
(„Kozak sequence“)
-
posible problems with splicing
(differences animal – plant)
Preparation of transgenic plant –
general procedure
1. One transformed cell
2. Multiplication (usually under selection) – callus formation
3. Induction of organogenesis (somatic embryogenesis)
- plant growth regulators
DNA insertion
selection
organogenesis
embryogenesis
transformed cell
transformed callus
Selection of transformed cells (plants)
Selection genes
resistances (degradation/modification of selection agens or
production of insensitive target)
- antibiotics (kanamycin, hygromycin)
- herbicides (Roundup® - glyphosate, Liberty®=basta –
glufosinate=phosphinotricin)
other, e.g. PMI (phosphomanose isomerase)
- conversion of manose-6-P na fru-6-P
Reporter genes
- visual selection (GFP, GUS, …)
Plant cell transformation methods
„natural“ method
• via Agrobacterium
– modifications (agroinfection, vacuum infiltration)
• with plant virus
– transient transformation (DNA not integrated)
– often primary infection with DNA copy of the genome (via
Agrobacterium)
biolistics („particle bombardment“, „microprojectile bombardment“)
„direct gene transfer“ to protoplast
• electroporation
• polyethylenglycol (PEG)
microinjection
Natural transformation with agrobacterium
(Agrobacterium tumefaciens)
• soil bacteria , G- (Rhizobiaceae), Ti plasmid
• „genetic parsitism“ in dicots
(external activation of monocots with acetosyringon)
• transfers several genes within T-DNA to plant cell
(causing tumor formation from transformed cell)
ipt – isopentenyl transferase
iaaH – indolacetamid hydrolase
genes for opine synthesis
Natural transformation with
agrobacterium
Disarmed agrobacterium
- tumor and opine genes removed
– only border sequences
necessary for T-DNA mobilization
- Ti plasmid splited into two
plasmids:
• T-DNA in small binary vector
• vir genes in helper plasmid
Border sequences:
- imperfect direct repeat 25 bp:
(LB a RB = right, left border)
- single strand breakage (virD2)
(D1/D2 dimer), strand replacement
Vir proteins in T-DNA transfer
induction of vir region expression
in reaction on phenolic compound
(VirA,VirG)
cleavage at T-DNA ends (VirD1,
VirD2)
porus formation (VirB1-11)
protection of ssT-DNA (VirE2)
transfer of T-DNA to nucleus
(VirE2 binds transcription factor
VIP, VirD2 contains NLS –
interaction with importin KAP-α)
targeted proteolysis of T-DNA
complex before integration (VirF)
Integration of T-DNA
- non-homologous recombination
- microhomology of inserted sequence in integration site
- often short deletions, rearangements, filler sequence
- RB more frequently preserved VirD2 protein)
Alternatively: formation of dsT-DNA, ligation into ds break of chromosome
Advantages of agro-transformation
• relatively high frequency of stable transformation
• low copy number (lower risk of RNAi induction)
• relatively long sequences (up to 45 kbp)
Transformation procedures:
• simple cocultivation of agro with plant tissue, cell
culture, ….
• vacuum infiltration of agro to the tissue
• inoculation in planta (flowers, leaves, …)
Potato transformation with agrobacterium
agrobacterium entrance
to the tissue
microscopical callus
agrobacterium
3-4 weeks
cocultivation
with injured
leaves
5-6 weeks
Floral dip in Arabidopsis thaliana
inoculation in planta
Inflorescences with buds into agrobacterium suspension
Floral dip in Arabidopsis thaliana
inoculation in planta
- target structure mostly the egg – forms
transformed embryo - plant
GUS reporter in developing transformed seeds
Agroinfiltration of tobacco
(in planta)
- preliminary tests of transgene expression
- without in vitro work
- cotransformed with viral
supressor of silencing (p19)
to reduce PTGS
+ P19
without P19
Biolistic method (Partickle gun, Gene gun)
• coating of Au or W particles with
DNA
• shot onto the tissue
Biolistics
over- or under-pressure to mobilize particles
Biolistics
• transformed cell through regeneration
• universal usage without limitations (only
regeneration ability)
• transformation of organelles (chloroplasts)
gold particle (1 m)
chloroplast (5 x 3 m)
Chloroplast transformation
- higher copy number per cell
- high protein levels
- without silencing
- targeting within plastom possible thanks to
active homologous recombination
- plastid genome usually missing in pollen
Disadvatages:
- no eukaryotic posttranslational modifications
- preparation of homoplastic and non-chimeric
plants is time consuming
Chloroplast transformation
Integration by recombination
– transgene surrounded with plastid sequences
Gradual selection of homoplasmic non-chimeric plants
Viral vectors
for transient biotechnological expression of proteins
- episomal
- non-integrated (= no position effect)
- high copy number
- strong expression – fast accumulation of product
- natural supressors of silencing (x PTGS)
- systemic spreading within plant
- often wide host spectrum
Viral vectors
- substitutional (e.g. for capsid protein, in
viruses with polyhedral capsids)
- insertional (possible in helical viruses)
- modular – splitting into more replicons
(e.g. TMV, Geminiviridae – polyhedral capsid)
Viral vectors
originally from Caulimoviridae
-
dsDNA genome – enabled sequence modification
low capacity (to 500 bp – polyhedral capsid)
polycistronic transcripts (complicated changes)
no practical application
at present:
-
-
helical viruses (TMV) tolerant to longer inserts
RNA viruses (enabled by discovery of RT – modification
of cDNA of viral genome)
primary infection in DNA form (agrobacterium) – viral
genome by transcription
Transgene incorporation
- ssT-DNA: non-homologous recombination
- dsDNA: reparation of DSB (double stranded break of DNA)
• simple joining of DNA ends
– blunt ends (frequent deletions)
– dominant in plants (most Eucaryots)
• homologous recombination
– integration in yeast, plastids and Physcomitrella
– „repair“ according to template (sister chromatid,
inserted sequence)
Homologous recombination – basic function
• crossing-over in meiosis (homologous chromosoms)
• DNA repair (sister chromatid)
Homologous recombination
1. extrachromosomal recombination - between two introduced
molecules - frequency in plants: 1 - 4 %
2. Intrachromosomal recombination - between two loci in the
same chromosome - frequency in plants: 10-5 až 10-6
3. gene targeting
recombination between introduced and chromosomal DNA
CHS lokus
(targeted integration of transgene
in yeast, plastids, Physcomitrella, …)
NPTII
in other eucaryots can be induced
by formation of DSB in the target
sequence!
vektor pro gene targeting
oblasti
homologie
upravený CHS lokus
NPTII
Frequency of homologous
recombination
(after insertion of homologous sequence)
ratio between homologous/non-homologous recombination
Higher plants 10-3 - 10-6
(high frequency of non-homologous recombination prevets homologous)
mammels
10-2 -10-5
lower eucaryots (yeast, protists, filamentous fungi)
> 10%
moss Physcomitrella patens
cca 90%
- effected by sequence length, ploidy, cell type, cell cycle phase, …
Stimulation of targeted DNA insertion
homologous recombination:
- DSB formation through site specific nuclease (ZFN, TALEN, CRISPR/Cas) –
integration by both homologous and non-homologous recombination
non-homologous recombination
- site specific rekombination systems of prokaryots and low eucaryots
(recombinase/recognized target site – has to be introduced to the genome in
advance)
Cre/lox
bacteriophage P1
Flp/frt
Saccharomyces cerevisiae
R/RS
Zygosaccharomyces rouxii
Cre/loxP
- Integration to previous insertion site
- Specific removal of selection marker gene
Targeted formation of DSB
Cleavage of unique genomic sequence that is recognized
- by chimeric nucleases, whose DNA binding domain
can be suited for specific sequence:
Zn-finger nucleases (ZFN)
TALEN (transcription activator-like effektor
nucleases
- by complementary RNA
CRISPR/Cas
Repair of DSB
Connected with DNA insertion
Without DNA insertion
- integrace transgenu s homologními
koncovými sekvencemi
(homologní rekombinací)
- autonomous repair often results in
local deletion
- integrace nehomologní
rekombinací v místě DSB
- presence (insertion) of DNA template
can direct specific site mutagenesis by
homologous recombination
Zn-finger domains – designed for specific sequence
DNA sequence recognition:
3(-4) nt/finger
- conserved aminoacids marked
- black circles – interaction with DNA
- místně specifické štěpení
- využití k cílené modifikaci DNA
(integraci transgenu)