Genetic engineering Sergiev P.V. 1755 Enzymes used for genetic - - PowerPoint PPT Presentation

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Genetic engineering Sergiev P.V. 1755 Enzymes used for genetic - - PowerPoint PPT Presentation

Nov 30, 2023 272 likes •1.01k views

MSU & SkolTech Genetic engineering Sergiev P.V. 1755 Enzymes used for genetic engineering Restriction endonucleases 5' 3' 3' 5' 5'p 3' 5' 3' 3' 5' 3' p ' 5 recognition sites usually symmetrical ( ) EcoRI G AATTC

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MSU & SkolTech

Genetic engineering

Sergiev P.V.

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Enzymes used for genetic engineering

Restriction endonucleases

5' 5' 3' 3' 5' 5' 3' 3' 5'p 3' p ' 5 3' recognition sites usually symmetrical EcoRI G AATTC CTTAA G GGG CCC G ACGTG after cleavage phosphate is located at ( ) 5' side SmaI CCC GGG PstI CTGCA G

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DNA ligase

Enzymes used for genetic engineering

5' 5' 3' 3' 5' 5' 3' 3' 5'p 3' p ' 5 3' T4 DNA ligase ATP , For ligation, phosphate should be at 5’-side

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DNA polymerases

Enzymes used for genetic engineering

DNA polymerases commonly used: Klenov fragment – medium polymerizing and 3’-exo activity T4 DNAP – strong polymerizing and 3’-exo activity Taq DNAP – thermostable polymerase, no 3’-exo activity (5’ exo) Pfu DNAP etc – thermostable polymerase, high 3’-exo activity

5' 5' 3' 3' 5' 3' DNA polymerase dATP,dCTP,dGTP,dTTP , 5' 3' 5' 5' 3' 3' 5' 3' DNA polymerase (exo) dATP,dCTP,dGTP,dTTP , 5' 3'

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Reverse transcriptase (revertase, RT)

Enzymes used for genetic engineering

5' 3' 5' 3' RT dATP,dCTP,dGTP,dTTP , 5' 3' RT commonly used: AMV RT – medium processivity, RNaseH MuMLV RT – high processivity, RNase H various engineered RT for cDNA synthesis

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RNA polymerase

Enzymes used for genetic engineering

5' 5' 3' 3' Т7 , RNA polymerase ATP,CTP,GTP,UTP 5' Т7 promoter 3' DNA RNA

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Polynucleotide kinase (PNK)

Enzymes used for genetic engineering

5' Т4 , PNK ATP 3' 5'p 3' 5' AP 3' 5'p 3'

Alkaline phosphatase (AP)

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Amidophosphite method

Chemical synthesis of DNA

O B O (MeO)2Tr O ... O B HO O ... O B O (MeO)2Tr O P N OMe N N N NH O B O (MeO)2Tr O P OMe O O B O ... O B O (MeO)2Tr O P O O B O ... OMe O

CF COOH

3

CH COOH, I , Py

3 2

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96 at a time

Chemical synthesis of DNA

Typical oligonucleotide 16-20 nt synthesis for several hours Up to 120 nt

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Genomic DNA Phenol deproteinization Ethanol precipitation

  • r binding to a column

Plasmid DNA Binding to a column

DNA purification from cells

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Polymerase chain reaction (PCR)

DNA amplificaton

5' 3' 5' 3' 5' 3' 5' 3' 5' 3' 5' 3' 5' 3' 5' 3' 5' 3' 5' 3' 5' 3' 5' 3'

1X 2X

96 oC, denaturation Specific temperature, hybridization 72 (or 68) oC, polymerization

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Polymerase chain reaction (PCR)

DNA amplificaton

Taq DNA polymerase – “simple”, quick, cheap, but many errors Pfu and derivatives – slower, expensive but low error frequency RT PCR – reverse transcription, followed by PCR

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DNA amplificaton

Quantitative PCR (qPCR)

1. ( ) Intercalating fluorophore SYBR green

  • 2. Taqman - separation of fluorophore and quencher
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Sanger sequencing

DNA (and RNA) sequencing

5' 5' 3' 3' ddA+dN ddC+dN ddG+dN ddT+dN C C G G G C G T G T G C A A T G C A T C A G C T G G C C C G C Ax G G C C C G C A C Ax G G C C C G C A C A C G T T Ax G G C C C G C A C A C G T T A C G T Ax G G C C C G C A C A C G T T A C G T A G T C G Ax

O P O O O- P O O- O P O O-

  • O

O B

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Detection of the primer extension products

DNA sequencing

ddA ddC ddG ddT

T A G C G T A C G T A T G G T G T T C C G A A C T G G

  • 1. Radioactive labeling of the 5’-end
  • 2. Radioactive labeling by a[32P]dNTP
  • 3. Fluorescent labeling of the 5’-end
  • 4. Fluorescent multcolor terminator

nucleotides

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Pyrosequencing (Roche 454)

Next generation sequencing

fragmentation conversion to blunt ends linker ligation purification on streptavidine beads

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Pyrosequencing (Roche 454)

Next generation sequencing

PCR mix hybridization suspension PCR streptavidin beads purification denaturation

  • ne bead - one type of DNA

(but multiple copies!)

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Pyrosequencing (Roche 454)

Next generation sequencing

“ ” 400 000 nano wells wells dNTP Ppi ATP свет dATP dGTP dCTP dTTP sulfurylase, luciferase Sequential feeding by dATP, dCTP dGTP,dTTP detection of light emission ,

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Pyrosequencing (Roche 454)

Next generation sequencing

Genome Sequencer FLX 1 000 000 400 reads ~ nt 10 1 000 000 000 hours ~ nt/day

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Illumina sequencing

Next generation sequencing

fragmentation linker ligation PCR conversion to blunt ends

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Next generation sequencing

denaturation hybridization Illumina sequencing

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Next generation sequencing

Illumina sequencing polymerization denaturation/hybridization

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Next generation sequencing

Illumina sequencing

polymerization denaturation bridge PCR restriction endonuclease

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Next generation sequencing

Illumina sequencing

DNA polymerase fluorescent 3'-azidomethyl dNTP ,

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Next generation sequencing

Illumina sequencing

scanning TCEP demodification next cycle

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Applications of NGS

Transcriptome (RNAseq) Genome wide binding sites of protein on DNA or RNA

cross-linking, fragmentation purification sequencing

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Applications of NGS

Translated regions (ribosome profiling)

fragmentation, ultracentrifugation sequencing mRNA

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Applications of NGS

chromosome conformation capture (3C) cross-linking, fragmentation sequencing, analysis

  • f hybrid reads

ligation

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Applications of NGS

Identification of DNA, associated with specific compartment (DamID) fragmentation purification sequencing in vivo modification Dam methylase, associated with specific nuclear compartment

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Vectors

PUC19.TXT

2686 bps

500 1000 1500 2000 2500

P-lac O-lac

HindIII SphI Sse8387 PstI BspMI SalI HincII AccI XbaI BamHI XmaI SmaI AvaI KpnI Asp718 SacI Ecl136 BanII EcoRI ApoI KasI EheI NarI BbeI NdeI AatII SspI XmnI ScaI GsuI Cfr10 BsaI Eam1105 AlwNI

  • ri

AflIII SapI

lacZ AmpR

Plasmid vector Usual features of a plasmid vector length up to 15 kbp

  • ri

selective marker (antibiotic resistance) (regulated) promoter polylinker

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Plasmid features

replication origins ColE1 (most frequently used) - pUC, pET, pQE etc /cell p15A (rather frequent) pACYC /cell pSC rare pKD /cell pCloDF13 exotic - pCDFDuet (Novagen) /cell pRSF exotic pRSFDuet (Novagen) /cell 40-100

  • 10-12

101 ( ) - 1-2 ( ) 20-40 1030 ( ) 100

Compatibility groups

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Plasmid features

Antibiotic resistance Ampicillin (most frequent) Kanamycin (frequent) Chloramphenicol (occasionally used) Tetracyclin (occasionally used) Streptomycin (rare) Spectinomycin (rare) Zeocine (rare) Promoters lac (IPTG) taq (IPTG) lacUV5 (IPTG) T5lac (IPTG) T7 (needs T7 RNAP) T7lac (IPTG, needs T7 RNAP) ara (arabinose) tet (anhydroteracyclin)

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Phage vectors

Vectors

M13

l

6 4 , 4 , . kbp up to kbp insert ssDNA in virions dsDNA in cells used for sequencing and mutagenesis 50 , 23 kbp up to kbp insert dsDNA used for cloning of large inserts

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Yeast vectors

Vectors

Centromere 2m high copy number 40-200 ARS 2m URA3 HIS3 LEU2 TRP1

  • ri ColE1

AmpR low copy number CEN 1-3 ARS + URA3 HIS3 LEU2 TRP1

  • ri ColE1

AmpR

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Methods of bacterial transformation

Chemical CaCl Ultracompetent cells MnCl growth at C heat shock + 18 +

2 2

  • Electroporation

kV needs low conductivity 1.4 - 2.8

  • +
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Methods for eukaryotes transformation

Yeast chemical transformation LiOAc + PEG High eukaryotes cation lipid electroporation viruses microinjection oocytes ( ) : ( )

O N

+

O

+ + + + + + + + +

  • +

+ + + + + + + + + +

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Host selection

Heterologous protein expression

  • E. coli
  • P. pastoris

mammalian cells S.cerevisiae baculovirus yield simplicity Post translation modification folding

++ + - ++ ++ ++ - --

  • + ++ ++
  • + ++ ++

± ± ± ±

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Datsenko-Wanner method

Genome manipulation in bacteria

KanR 5'-flank 3'-flank gene of interst PCR-product KanR l a b g RED proteins ( ) ' exonuclease ( ) ssDNA binding strand annealing protein ( ) RecBCD inhibitor

  • 5

, , electroporation abg 40 bp 40 bp

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Integration by homologous recombination

Genome manipulation in yeast

>40 bp 5'-flank 3'-flank gene yeast tarnsformation homologous recombination >40 bp marker marker

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Transient transfection 48-72 hours plasmid vector expressed protein

Methods for expression in eukaryotes

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Methods for expression in eukaryotes

plasmid vector genes necessary for virus packaging viral particles with the gene

  • f interest

Stable cell line construction

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Stable cell line construction

Methods for expression in eukaryotes

lentivirus vector DNA intergated to the genome

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siRNA

Inactivation of expression

gene mRNA mRNA siRNA mRNA mRNA degradation

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shRNA

Inactivation of expression

gene mRNA mRNA mRNA mRNA degradation shRNA

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Transposase assisted knock-in

Sleeping beauty system insert flanked by IR transposase expression vector

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Создание мышей с измененным геномом

Knock-in: вставка гена а случайное место генома Knock-out: инактивация гена редактирование гена

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Зачем ?

Для создания моделей генетических заболеваний

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Зачем?

Чтобы разобраться, как это устроено

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Knock-out: инактивация гена использование гомологичной рекомбинации для редактирования генома Нужны очень большие фланкирующие области: плазмиды не подходят, нужны БАКи создание – рекомбиниринг в E. coli >10 000 п.о. >10 000 п.о.

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Knock-out: инактивация гена использование гомологичной рекомбинации для редактирования генома ~800 п.о. ~800 п.о. Достаточны плазмиды, для микромутаций даже олигонуклеотиды с фланками 60-90 нт.

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Knock-out: инактивация гена Разрыв может привести к микроделециям и сбою рамки считывания

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CRISPR (clustered regularly interspaced short palindromic repeats

R Cas genes R R R R R R spacers

Спейсеры содержат последовательности ДНК фагов CRISPR-Cas система делает бактерию иммунной к фагам, которые она «знает»

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R Cas гены R R R R R R Cas белки РНК транскрипт процессированная крРНК Cascade комплекс

CRISPR (clustered regularly interspaced short palindromic repeats

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CRISPR-Cas система делает бактерию иммунной к фагам, которые она «знает»

CRISPR (clustered regularly interspaced short palindromic repeats

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Cascade узнает и разрезает ДНК CRISPR

PAM протоспейсер Cascade узнавание протоспейсера Разрезание мишени Деградация мишени

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Почему CRISPR лучше?

Nat Protoc. 2013 November ; 8(11): 2281

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Почему CRISPR лучше?

Nat Protoc. 2013 November ; 8(11): 2281

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Как это происходит

METTL3 D19

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Как делать трансгенных мышей? модифицировать стволовые клетки модифицировать зиготы

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Как делать трансгенных мышей? Мозаичные мыши

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А если мутация летальна?

loxP loxP

X

Cre loxP loxP Cre loxP

Ген (экзон гена) Ген (экзон гена)

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Как индуцировать Cre рекомбиназу? Работа эстрогенового рецептора эстроген

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Как индуцировать Cre рекомбиназу? тамоксифен

loxP loxP Cre Cre Cre

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Стволовые клетки Есть доступные линии, большинство агути 129, иногда C57BL6 A/a бластоциста ICM Без поддерживающих клеток На поддерживающих клетках (MEF) Специальная среда с ингибиторами дифференцировки (LIF, 2i)

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Вектора для работы со стволовыми клетками

Skarnes WC, et al., Nature. 474, 337-342.

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Как, собственно делать трансгенных мышей? Вазэктомированный самец Самка рецепиент

♀ ♀ ♂ ♂

Стволовые клетки

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Инъекции в зиготу Чего инъекции?

  • 1. Плазмида с CRISPR/Cas9 в пронуклеус
  • 2. CRISPR РНК+Cas9 мРНК в цитоплазму
  • 3. CRISPR РНК+Cas9 мРНК+ДНК для гомологичной

рекомбинации в пронуклеус (можно добавлять ингибитор лигазы)

  • 4. ДНК для knock-in в пронуклеус
  • 5. Лентивирусы за блистерную оболочку или

транспозон и транспозаза

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Как, собственно делать трансгенных мышей? Вазэктомированный самец Самка рецепиент

♀ ♀ ♂ ♂

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Инъекции в зиготу

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Инъекции в зиготу Какая эффективность? Или несколько цифр для прикидки Из 10 доноров яйцеклеток 3-9 покрыты, ~100 яйцеклеток Живых яйцеклеток после инъекции 70-80% (10-40%) Развиваются до бластоцист 25-65% (лучше с РНК) Рождаются из пересаженных 8-25% Трансгенов (knock-out) из рожденных 25-100% Трансгенов (замена) из рожденных ~5% (25% с ингибитором)

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КОНЕЦ