Transcript Diapositiva 1 - Cayetano Heredia University

Reverse
Genetics
J. Lieb
Mouse Embryos
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:
• 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.
Review of Last Lecture
"Model" Organisms in Biology
What allows us to use them?
1. All organisms share similar cellular machinery
2. 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
Review of Last Lecture
Transgenics
Using 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.
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
GENE TARGETTING
• Dirigido a un gen específico.
• Silenciamiento del gen a nivel del RNA.
• Reemplazo o modificación del gen.
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
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.
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´.
mRNA de histonas dependientes de replicación
El oligo Antisense
puede estar dirigido a:
- bloquear la formación
del stem-loop
- bloquear la interacción
del mRNA con el
snRNA.
El pre-mRNA de HIV integrado debe formar 2 hairpins
(TAR) necesarios para la unión de la proteína TAT al RNA
Antisense dirigido a bloquear
la formación de los hairpins
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
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
Mecanismo de corte por ribozima
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’
A
C U
I
G
A
A
U
G
AG
CG
GC
GC
CG
GC
G U
CU
Ribozima
TRANSGENIA
Conceptos
Diferencia entre clonación y transgenia
WHY?
•Identification of gene function
•Generation of animal models of diseases
•Drug validation
•Cell and organ research
Others…
How many genes are there in mammalian cells?
E. coli
S. cerevisiae
D. melanogaster
C. elegans
H. Sapiens
4.6 Mb
13.5 Mb
165 Mb
97 Mb
3,300 Mb
4,288 genes
6,034 genes
12,000 genes
19,099 genes
40,000 genes
Genome project was completed in 2002 (still regions that are unclear)
Genomics and proteomics
Gene expression profiling
Phenotypes
(Exon profiling)
Studied on the mechanism
of gene expression.
Transgenic technologies
MAMMALIAN EMBRYO MANIPULATIONS
Animal models?
In theory, all mammalian embryos can be used.
Mouse and rats
Research
Farm animals
Commercial uses:
Human
Improve health, Cure diseases , others?
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
MAMMALIAN EMBRYO MANIPULATIONS
NON-TRANSGENIC
No modifications to the genome.
TRANSGENIC
Modifications to the genome.
Germline mutations
Somatic cell mutations
Transgenic and embryo manipulation in mammals
2
1
Transgenic Animals by
pronucleus injection
Natural or
in vitro
fertilization
3
-Genotyping of genetic diseases
-Freezing and storing
-Embryo cleavage and cloning
Spermatozoide
Morula
Oocyte
Enucleation
Nuclear transfer
and cloning
8
4
Stem cells
Cell
therapy
7
5
Neuronal cells
- Isolaton of embryonic stem cells
- Gene targeting
in vitro
Différentiation
Muscle cells
Blastocyst
Epithelial cells
6
Primary cells
culture
Oocyte
Fertilized
egg
Morula
Zygote
6hrs
18hrs
36hrs
Blastocyst
Implantation
4 days
3 days
48hrs
Oocyte
-Nuclear
Transfer
-Cloning
Fertilized
egg
Blastocyst
Morula
Freezing Splitting Genotyping Infection
Transgenic
By Microinjection
-Embryonic stem
Cells
-Gene targeting
Nuclear Transfer Technology
For cloning
Oocyte
Nucleus of stem cells
or others
Nuclear Transfer Technology
Usage?
Cloning of:
- Valuable cheptel (farm animals)
- Endangered species
- Basic research in stem cell tech.
- Others?
Gene Locus: Includes both the promoter and the transcription unit!
Distal
Proximal
>100kb
Approx. 1kb
Promoter
Coding and uncoding sequences
from 1kb to >200kb
Transcription
Unit
Transgenic Technology
Promoter
Intron
cDNA
AAAA
Temporal and spatial
expression
Gene of interest
AAAA
Transgene
<1kb to >200kb
Transgenic Technology
Promoter
cDNA
1- Tissue specific (brain, liver, muscle …)
2- Ubiquitous
3- Inducible (tetracyclin, interferon...
Gain of Function
- wild-type gene
- mutant
Loss of Function
- dominant negative
- antisense
- ribozyme
Identification of the important features of a promoter.
A: Comparison of sequences: Increasing uses
Databases: Genebank and others.
B: Use of reporter genes:
Promoter of gene x
Promoter of gene x
Gene x
Reporter gene
- B galactosidase
- Luciferase
- GFP
- others….
Transgenic Technology
DNA
6hrs
Mosaic
Transgenic Technology
Fos
X
X
Ste.
CD1
FVB/N
Fos
6hrs
Reimplantation
in the oviduct
Fos
Pregnancy
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
Embryonic stem Cells
and
Gene targeting
Gene knock-out and Gene Knock-in
Knock-out Mice
Embryonic Stem cells
Homologous recombination
Oliver Smithties
Mario Capecchi
Neomycin
Episomal
In chromatin
Neomy
X
mycin
Neomy
X
mycin
Approx. 500 bp
Homologous recombination
1- Length of homologous sequences
Hom. Rec. Efficiency
Base pair
25bp
2000bp
2- Isogenic DNA
Gene targeting
X
X
NEOMYCIN
NEOMYCIN
Total 4 Kbp (each arm not less than 500bp)
Delete coding sequences
Change reading frame
Transcription of Neo in antisense direction
Embryonic Stem cells
Blastocyst
3 days
C57Bl/6
Black
129/sv
(agouti)
Inner Cell Mass
1-totipotent
Foster mother
2 day-pregnant
2-tissue culture
3-Transfectable
4-Selection
5- Differentiation
In vitro
X
X
1 WT
2 Hetero
1 Homo
Site specific recombination: Cre-Lox system, Flp recombination
From P1 phage
Excise and integrate DNA
Cre recombinase
Excise
a
b
a
c
b
a
Integrate
e
c
c
b
Lox site
(approx 30 bp)
g
f
e
f
g
Lox mouse
Cre lox in the mouse
X
Cre mouse
KO in the brain only
KO in the adult only
Brain
Adult-P
- Temporal and spatial targeting
- knock-in
- single point mutation
- translocation
What about
transgenics in
mammals?
Day 50
(end of Week 7)
A relatively new (1980s)- molecular approach
Recipe for a gene "knockout“ in mice:
Step 1
Problem: 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
ES or Embryonic
stem cells:
Blastocyst-stage cells
that have been coaxed
and coddled into
growing in culture
Blastocyst stage cells can be easily incorporated into a different
blastocyst stage embryo, leading to production of chimeras
A mouse with
“3 parents”
Adding a gene: Production of Transgenic Mice
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
Production of Transgenic Mice 3
Production of Transgenic Mice 3
OK, we've added a gene
(Transgenics).
Now, we want to make a KO
(Reverse Genetics)
Recipe to "knockout" a gene:
Step 2
A normal cell has two copies of gene X:
mRNA
Gene X
mRNA
Mario Capecchi
Gene X
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.
Neo resistance gene
No
mRNA
Gene X
mRNA
Gene X
Recombinant ES
cells can then be
selected in culture.
Oliver Smithies
(UNC)
Technique for Gene Targeting (1 of 3)
Technique for Gene Targeting (2 of 3)
Technique for Gene Targeting (3 of 3)
Morphological Analysis of Bmp7
Knockout Mice
Morphological Analysis of Bmp7
Knockout Mice
Mouse models of human disease
help us to design and test new treatments
www.hgu.mrc.ac.uk/Research/Devgen/Cysfib/julia.htm
www.cf.ac.uk/biosi/staff/jacob/teaching/ionchan/cftr.jpg
A transgenic human
Treated for SCID
Other Reverse Genetic Approaches
• Site-directed mutagenesis
• RNAi
• Chemicals (Chemical Genetics)
Site-directed mutagenesis
Gene Replacement
RNA Interference
Method 1
Method 2
Method 3
Mechanism of RNAi
iRNA
RNA de interferencia
RISC: RNA induced
silencing complex
RNAi en la expresión de
GFP
Fig 4. Small interfering RNAs vs Small temporal RNAs
Forward and Reverse
"Chemical 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.
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-
Ejemplo: knock-out del gen BMP-7
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
en presencia de Neomicina
sobrevive
• Gen Timidina Kinasa del HSV (marcador de selección negativa)
Cell + TK HSV
en presencia de ganciclovir
muere