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Reverse Genetics

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Reverse Genetics. Mouse Embryos. J. Lieb April 19, 2006. Wild-type. Bmp7 -/-. Remember Forward Genetics? : Phenotype  Gene or Mutations First  Molecular Analysis Second. Reverse Genetics Gene  Phenotype or Molecular Analysis First  Mutations Second. Example Uses: - PowerPoint PPT Presentation
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Reverse Genetics J. Lieb April 19, 2006 Wild-type Bmp7 -/- Mouse Embryos
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Page 1: Reverse Genetics

Reverse Genetics

J. Lieb

April 19, 2006

Wild-type Bmp7 -/-

Mouse Embryos

Page 2: Reverse Genetics

Remember Forward Genetics? :

Phenotype Gene

or

Mutations First Molecular Analysis Second

Page 3: Reverse Genetics

Reverse Genetics

Gene Phenotypeor

Molecular Analysis First Mutations Second

Example Uses:

• Understand the function of a gene homolog characterized in another organism

• Understand the function of individual amino acids or protein domains.

• Create “conditional alleles” of a gene.

Page 4: Reverse Genetics

Review of Last Lecture

"Model" Organisms in Biology

What allows us to use them?1. All organisms share similar cellular machinery2. All animals use this machinery in similar ways to direct embryonic

development

Why use them?Perform controlled experiments on a large number of samples to

learn about:• Basic Molecular Mechanisms (Yeast Cell Cycle: Cancer)• Mechanisms of Genetics (Mendel’s Peas; Fruit Flies)• Embryonic Development (C. elegans, Sea Urchins; Mice)

What does one look for when choosing a model?

Fast, cheap, easy to observe and manipulate, and has the feature you want to study

Page 5: Reverse Genetics

TransgenicsUsing the power of molecular biology to isolate and clone the DNA of our choice, and then to express it in a controlled manner in the organism of choice.

Review of Last Lecture

Why?

• To study the role of a gene in development

• To see where a gene is expressed

• To understand what happens when a gene is misregulated

• To “cover” a genetic defect

Page 6: Reverse Genetics

GENE TARGETTING

• Dirigido a un gen específico.

• Silenciamiento del gen a nivel del RNA.

• Reemplazo o modificación del gen.

Page 7: Reverse Genetics

SILENCIAMIENTO DEL GEN

• Acción a nivel de mRNA (evitando que sea traducido)

Mecanismos:

• Oligonucleótidos Antisentido : oligonucleótido con la secuencia complementaria al mRNA blanco (target )

• Ribozimas: RNAs con actividad catalítica, se une y corta al mRNA blanco (target)

• iRNA : RNA de interferencia

Page 8: Reverse Genetics

Oligonucleótidos antisentido (Antisense oligos)

• DNA o RNA de hebra simple

• Secuencia complementaria al mRNA target

• Mecanismo de acción : formación de doble cadena y:

Bloqueo del Inicio / elongación de traducción

Alteración del procesamiento del mRNA : si está dirigido a la secuencia limite exón/intrón bloquea la función de los snRNAs que intervienen durante el splicing

Alteración de la estabilidad / vida media del mRNA: si el oligo está dirigido a una secuencia en el 3´ UTR y que impida la formación de hairpins propios del mRNA que lo estabilizen; por ejemplo: mRNAs de histonas no tienen poly A, pero sí forman hairpins en el 3´ UTR.

Page 9: Reverse Genetics

En vertebrados existen dos tipos de histonas:

- Histonas independientes de replicación : sus mRNAs son polyadenilados

- Histonas dependientes de replicación, sus mRNAs: - presentan cola de polyA - presentan una estructura de tipo stem-loop en el 3´UTR - presenta una secuencia de reconocimiento para el procesamiento o maduración del extremo 3´.

Page 10: Reverse Genetics

El oligo Antisense puede estar dirigido a:

- bloquear la formación del stem-loop

- bloquear la interacción del mRNA con el snRNA.

mRNA de histonas dependientes de replicación

Page 11: Reverse Genetics

Antisense dirigido a bloquear la formación de los hairpins

El pre-mRNA de HIV integrado debe formar 2 hairpins (TAR) necesarios para la unión de la proteína TAT al RNA

Page 12: Reverse Genetics

Oligonucleótidos con mayor tiempo de vida media

PNAs : Peptide-nucleic acids

• Esqueleto formado por enlaces peptídicos : estabilidad, mayor tiempo de vida media

• Bases nitrogenadas : confieren especificidad de acción

Principal desventaja :

- toxicidad

Page 13: Reverse Genetics

RIBOZIMAS

• RNAs con actividad catalítica, se une y corta al mRNA blanco (target)

• Estructura estable : hammerhead

• Pueden ser quimicamente modificados los extremos para incrementar su vida media

Page 14: Reverse Genetics

5’- - c g g a g u c a c u u c g - - 3’ mRNA 3’ G C C U C A U G A A G C 5’

AIAGCGGCG

GCCGC

UC

C

G U

AGU

AG

U

Ribozima

Mecanismo de corte por ribozima

Page 15: Reverse Genetics

TRANSGENIA

Conceptos

Diferencia entre clonación y transgenia

Page 16: Reverse Genetics

•Identification of gene function

•Generation of animal models of diseases

•Drug validation

•Cell and organ research

Others…

WHY?

Page 17: Reverse Genetics

How many genes are there in mammalian cells?

E. coli 4.6 Mb 4,288 genesS. cerevisiae 13.5 Mb 6,034 genesD. melanogaster 165 Mb 12,000 genesC. elegans 97 Mb 19,099 genesH. Sapiens 3,300 Mb 40,000 genes

Genome project was completed in 2002 (still regions that are unclear)

Gene expression profiling (Exon profiling)

Phenotypes

Studied on the mechanismof gene expression.

Genomics and proteomics

Transgenic technologies

Page 18: Reverse Genetics

MAMMALIAN EMBRYO MANIPULATIONS

Animal models?

Mouse and rats Research

Commercial uses:Farm animals

Improve health, Cure diseases , others?

In theory, all mammalian embryos can be used.

Human

Page 19: Reverse Genetics

The MOUSE Life span: approx. 2.5 years

Gestation : 21 days

Litter size: 8 to 12

Generation time: three months

Several inbred and outbred strains

Genomic database

Most advance genetic technologies

Cost per mouse/$2 to 24$

Housing cost

Over 90% identical to human genome

Large enough for physiological studies

Page 20: Reverse Genetics

MAMMALIAN EMBRYO MANIPULATIONS

NON-TRANSGENIC

TRANSGENIC

No modifications to the genome.

Modifications to the genome.

Germline mutations Somatic cell mutations

Page 21: Reverse Genetics

Natural or in vitro

fertilization

Spermatozoide

Oocyte

Enucleation

Nuclear transfer and cloning

Transgenic Animals by pronucleus injection

-Genotyping of genetic diseases -Freezing and storing -Embryo cleavage and cloning

Transgenic and embryo manipulation in mammals

Morula

Blastocyst

- Isolaton of embryonic stem cells - Gene targeting

Primary cells culture

Cell therapy Neuronal cells

Muscle cells

Epithelial cells

Stem cells

1

23

4

in vitro Différentiation

5

6

7

8

Page 22: Reverse Genetics

Spermatozoide

Oocyte

6hrs 18hrs 36hrs 48hrs

Fertilizedegg

ZygoteMorula

Blastocyst

3 daysImplantation

4 days

Page 23: Reverse Genetics

Spermatozoide

Oocyte Fertilizedegg Morula Blastocyst

-NuclearTransfer-Cloning

Transgenic By Microinjection

Freezing Splitting InfectionGenotyping

-Embryonic stemCells

-Gene targeting

Page 24: Reverse Genetics

Oocyte

For cloning

Nuclear Transfer Technology

Nucleus of stem cells or others

Page 25: Reverse Genetics

Nuclear Transfer Technology

Usage?

Cloning of: - Valuable cheptel (farm animals) - Endangered species- Basic research in stem cell tech.- Others?

Page 26: Reverse Genetics

Distal>100kb

ProximalApprox. 1kb

Promoter TranscriptionUnit

Coding and uncoding sequencesfrom 1kb to >200kb

Gene Locus: Includes both the promoter and the transcription unit!

Page 27: Reverse Genetics

Transgenic Technology

Promoter

Temporal and spatialexpression

cDNA Intron

Gene of interest

AAAA

AAAA

Transgene

<1kb to >200kb

Page 28: Reverse Genetics

Transgenic Technology

Promoter

cDNAGain of Function

1- Tissue specific (brain, liver, muscle …)2- Ubiquitous

3- Inducible (tetracyclin, interferon...

Loss of Function

- wild-type gene- mutant

- dominant negative- antisense- ribozyme

Page 29: Reverse Genetics

Identification of the important features of a promoter.

A: Comparison of sequences: Increasing uses

B: Use of reporter genes:

Databases: Genebank and others.

Gene xPromoter of gene x

Promoter of gene x Reporter gene - B galactosidase- Luciferase- GFP- others….

Page 30: Reverse Genetics

Transgenic Technology

6hrs

MosaicDNA

Page 31: Reverse Genetics

Transgenic Technology

6hrs

FVB/N

XX

Ste.

CD1

Fos

Fos

Reimplantationin the oviduct

Pregnancy

Fos

Page 32: Reverse Genetics

Transgenic Technology

Advantages Disadvantages

- Short time to produce(21 days)

- High level of expression- Cheaper cost- Simple vectors

- Random integration- Multiple integration sites- Each animal has

different genotype

Page 33: Reverse Genetics

Embryonic stem Cellsand

Gene targeting

Gene knock-out and Gene Knock-in

Page 34: Reverse Genetics

Knock-out Mice

Homologous recombination

Embryonic Stem cells

Oliver SmithtiesMario Capecchi

Neomycin

Neomy

mycinX

Approx. 500 bp

Neomy

mycinX

In chromatin Episomal

Page 35: Reverse Genetics

Homologous recombination

1- Length of homologous sequences

Hom. Rec. Efficiency

Base pair

25bp 2000bp

2- Isogenic DNA

Page 36: Reverse Genetics

NEOMYCIN

X X

Gene targeting

NEOMYCIN

Total 4 Kbp (each arm not less than 500bp)Delete coding sequencesChange reading frame

Transcription of Neo in antisense direction

Page 37: Reverse Genetics

Embryonic Stem cells Blastocyst 3 days

Inner Cell Mass

1-totipotent

2-tissue culture

3-Transfectable

4-Selection

5- DifferentiationIn vitro

129/sv(agouti)

C57Bl/6Black

Foster mother2 day-pregnant

Page 38: Reverse Genetics

X

X

1 WT 2 Hetero 1 Homo

Page 39: Reverse Genetics

Site specific recombination: Cre-Lox system, Flp recombination

From P1 phage

Cre recombinase Lox site(approx 30 bp)

Excise and integrate DNA

Excisea b c

a bc

a c

b

Integratege

f

e f g

Page 40: Reverse Genetics

Cre lox in the mouse

- Temporal and spatial targeting- knock-in- single point mutation- translocation

Brain

KO in the brain only

KO in the adult only

Adult-P

X

Lox mouse

Cre mouse

Page 41: Reverse Genetics

Day 50 (end of Week 7)

What about transgenics in mammals?

Page 42: Reverse Genetics

A relatively new (1980s)- molecular approachRecipe for a gene "knockout“ in mice:

Step 1Problem: Find a cell line that can grow in tissue culture but also retains the potential to become part of a real embryo.

Solution- Embryonic stem cells

Page 43: Reverse Genetics

ES or Embryonic stem cells:

Blastocyst-stage cellsthat have been coaxedand coddled intogrowing in culture

Page 44: Reverse Genetics

Blastocyst stage cells can be easily incorporated into a different blastocyst stage embryo, leading to production of chimeras

Page 45: Reverse Genetics

A mouse with“3 parents”

Page 46: Reverse Genetics

Adding a gene: Production of Transgenic Mice

Page 47: Reverse Genetics

Production of Transgenic Mice 2

Embryonic stem cells (ES cells) are then incorporated into blastocysts, with the hope that they “go germline”.

If so, a line is created

Page 48: Reverse Genetics

Production of Transgenic Mice 3

Page 49: Reverse Genetics

Production of Transgenic Mice 3

Page 50: Reverse Genetics

OK, we've added a gene (Transgenics).

Now, we want to make a KO(Reverse Genetics)

Page 51: Reverse Genetics

Mario Capecchi

mRNA

Gene X

mRNA

Gene X

A normal cell has two copies of gene X:

No mRNA

Gene X

mRNA

Gene X

Neo resistance gene

Scientists use homologous recombination to insert gene for resistance to the drug neomycin into the middle of one of the copies of gene X, destroying its function.

Recipe to "knockout" a gene: Step 2

Oliver Smithies(UNC)

Recombinant ES cells can then be selected in culture.

Page 52: Reverse Genetics

Technique for Gene Targeting (1 of 3)

Page 53: Reverse Genetics

Technique for Gene Targeting (2 of 3)

Page 54: Reverse Genetics

Technique for Gene Targeting (3 of 3)

Page 55: Reverse Genetics

Morphological Analysis of Bmp7 Knockout Mice

Page 56: Reverse Genetics

Morphological Analysis of Bmp7 Knockout Mice

Page 57: Reverse Genetics

www.hgu.mrc.ac.uk/Research/Devgen/Cysfib/julia.htm www.cf.ac.uk/biosi/staff/jacob/teaching/ionchan/cftr.jpg

Mouse models of human diseasehelp us to design and test new treatments

Page 58: Reverse Genetics

A transgenic humanTreated for SCID

Page 59: Reverse Genetics

Other Reverse Genetic Approaches

• Site-directed mutagenesis

• RNAi

• Chemicals (Chemical Genetics)

Page 60: Reverse Genetics

Site-directed mutagenesis

Page 61: Reverse Genetics

Gene Replacement

Page 62: Reverse Genetics

RNA Interference

Method 1 Method 2 Method 3

Page 63: Reverse Genetics

Mechanism of RNAi

Page 64: Reverse Genetics

iRNA RNA de interferencia

RISC: RNA induced silencing complex

Page 65: Reverse Genetics

RNAi en la expresión de GFP

Page 66: Reverse Genetics
Page 67: Reverse Genetics
Page 68: Reverse Genetics
Page 69: Reverse Genetics
Page 70: Reverse Genetics
Page 71: Reverse Genetics

Fig 4. Small interfering RNAs vs Small temporal RNAs

Page 72: Reverse Genetics
Page 73: Reverse Genetics
Page 74: Reverse Genetics

Forward and Reverse "Chemical Genetics"

Page 75: Reverse Genetics

REEMPLAZO DE GENES

• Integración del fragmento de DNA en el genoma del hospedero- en el sitio del gen homólogo o en sitios al azar. La integración en sitios al azar es más frecuente.

• Si existen secuencias homólogas en el genoma puede haber recombinación homóloga. Se da el reemplazo del gen específico del hospedero (Knock-out).

• En la integración al azar, la expresión del gen puede verse afectada según el lugar de inserción, puede interrumpir o afectar a otros genes.

• Células somáticas vs. Células germinales - transgénico.

Page 76: Reverse Genetics

GENERACION DE ANIMALES TRANSGENICOS

1. Cultivo in vitro de células troncales de embrión (Embryonic stem- ES).

2. Preparación del gen a insertar. (Ej: BMP7 clonado, es interrumpido por el gen de resistencia a Neomicina como marcador de selección).

3. Transferencia del DNA exógeno a las células.

4. Selección de las células donde ha ocurrido el reemplazo del gen utilizando el marcador de selección (resistencia a Neomicina)

5. Las células seleccionadas se insertan en un embrión nuevo, el cual es colocado al útero.

6. Progenie resultante: quimeras con algunos tejidos heterocigotes y otros tejidos silvestres.

7. El cruce de una quimera con un animal silvestre dará una progenie heterocigote

(BMP7+/BMP7-) si las cells ES modificadas han contribuido a la linea germinal.

8. Del cruce entre los heterocigotes, aprox. 25% de la progenie será homocigote

trangénico BMP7- /BMP7-

Page 77: Reverse Genetics

Ejemplo: knock-out del gen BMP-7

Page 78: Reverse Genetics
Page 79: Reverse Genetics

MARCADORES DE SELECCIÓN

• Selección positiva : célula + marcador insertado la célula vive

• Selección negativa: célula + marcador insertado la célula muere

Por ej:

• Gen de resistencia a Neomicina (marcador de selección positiva)

Cell + Neor sobrevive

• Gen Timidina Kinasa del HSV (marcador de selección negativa)

Cell + TK HSV muere

en presencia de Neomicina

en presencia de ganciclovir


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