Transgenosismethods of transformation

• transient Foreign DNA introduction

• stable DNA is / is not integrated to chromosom/plastom/chondriom)

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:

TERMINATOR - balanced with promoter! - start site and direction of transcription- interacts with trans elements (transcription factors = specific spatio-

temporal regulation of activity – specific host promoters)- special promoters: constitutive, inducible (EtOH, heat, estradiol)- origin: plants, plant pathogenes, synthetic

Translation

- codon usage can differ (tRNA frequency)- problematic if expressing plantgene in bacteria

- ATG sequence context – important!!!

(„Kozak sequence“)

- posible problems with splicing(differences animal – plant)

AACAATG Stop

coding sequencemRNA

selection

Preparation of transgenic plant – general procedure

1. One transformed cell

transformed cell

organogenesis

embryogenesis

3. Induction of organogenesis (somatic embryogenesis)- plant growth regulators

DNA insertion

2. Multiplication (usually under selection) – callus formation

transformed callus

Selection genesresistances (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, …)

Selection of transformed cells (plants)

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

- 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

Disarmed agrobacterium

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

cocultivationwith injured leaves

agrobacterium

agrobacterium entranceto the tissue

3-4 weeks

5-6 weeks

microscopical callus

Floral dip in Arabidopsis thaliana inoculation in planta

Inflorescences with buds into agrobacterium suspension

- target structure mostly the egg – forms transformed embryo - plant

GUS reporter in developing transformed seeds

Floral dip in Arabidopsis thaliana inoculation in planta

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

Homologous recombination – basic function • crossing-over in meiosis (homologous chromosoms)• DNA repair (sister chromatid)

• 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)

Transgene incorporation - ssT-DNA: non-homologous recombination - dsDNA: reparation of DSB (double stranded break of DNA)

Homologous recombination1. 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(targeted integration of transgenein yeast, plastids, Physcomitrella, …)

in other eucaryots can be inducedby formation of DSB in the target sequence!

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

Connected with DNA insertion

- integrace transgenu s homologními koncovými sekvencemi(homologní rekombinací)

- integrace nehomologní rekombinací v místě DSB

Without DNA insertion- autonomous repair often results in local deletion

- presence (insertion) of DNA template can direct specific site mutagenesis by homologous recombination

Repair of DSB

Zn-finger domains – designed for specific sequence

DNA sequence recognition:

3(-4) nt/finger

- místně specifické štěpení- využití k cílené modifikaci DNA(integraci transgenu)

- conserved aminoacids marked- black circles – interaction with DNA