gene targeting and gene trapping comaprison

gene targeting and gene trapping comaprison

1. GENE TRAPPING
Paras Yadav*, Jaspreet Singh Arora*, Sachinandan
De*, Tirtha Kumar Datta*, Surender Lal Goswami*,
Aarti Bhardwaj
$
, Shalini Jain
#
and Hariom Yadav
#
*Animal Biotechnology,
#
Animal Biochemistry Division, National
Dairy Research Institute, Karnal-132001, Haryana, India
$
Meerut Institute of Engeenering and Technology, Meerut, U.P.,
India

2. Gene trapping is a form of insertional mutagenesis
specifically designed to disrupt gene function by
producing intragenic integration events.
Evans, M.J. (1998) Evans, M.J. (1998) Dev. Dyn.Dev. Dyn., , 212212, 167-169 , 167-169
What is gene trapping ?What is gene trapping ?

3. • A random integration of a reporter gene
construct, called entrapment vector into
genome.
•Productive integration events bring the
reporter gene under the transcriptional
regulation of an endogenous gene.

4. Promoter a combination of short sequence elements to which
RNA polymerase binds in order to initiate transcription of a gene.
Enhancer a set of short sequence elements which stimulate
transcription of a gene.
Polyadenylation addition of typically 200 A residues to the
3' end of a mRNA. The poly(A) tail is important for stabilizing
mRNA.
Regulatory Components of a Gene – Regulatory Components of a Gene –
important for its expressionimportant for its expression

5. Basic Strategy in Gene TrapBasic Strategy in Gene Trap

6. Gene trap StrategyGene trap Strategy
Choosing proper vector and delivery system
Selecting the clones with markers
Identification of location of the insert in the clone
Studying biological questions: Production of chimeras

7. Components of gene trap:Components of gene trap:
•Mouse or human embryonic stem cell (for
mammalian model)
•Entrapment vector construct having the
reporter gene and selectable marker.

8. Reporter genesReporter genes
•The E.coli lacZ gene
•The E.coli. Chloramphenicol acetyltransferase (CAT)
gene
•The firefly luciferase gene
•The jelly fish green flourecence protein (GFP) gene

9. Selectable MarkersSelectable Markers
Positive selection:
•Neomycin phosphotransferase gene (neo
R
)
•Puromycin selection (Puro)
Negative selection:
•Herpes Simplex Thymidine kinase gene (hsv-tk)
•Diphtheria toxin gene

10. Types of vectorsTypes of vectors
•Enhancer trap vector
•Promoter trap vector
•Gene trap
•poly A trap

11. promoter
Enhancer
DNA
RNA
protein

proteinXproteinX β-gal Neo
R
promoter
lac Z
P'
pApA
Endogenous gene X
Vector Integration
Vector
neo
lac Z
neo
Enhancer TrapEnhancer Trap
lac Z neo
Exon 1 Exon 2 Exon 3

12. Endogenous gene X
lac Z
pA
P'
neo
pA
Vector Integration
DNA
RNA
protein

proteinXproteinX β-gal Neo
R
lac Z
neo
Promoter TrapPromoter Trap
lac Z neo

13. SA
Endogenous gene X
SA
lac Z
pA
P'
neo
pA
Vector Integration
DNA
RNA
protein
Spliced transcript

proteinXproteinX
β-gal Neo
R
Gene TrapGene Trap
lac Z
neo
SA lac Z
neo

14. SA pA
P'
neo
SD
lac Z
Vector Integration
pA
pA

proteinXproteinX
β-gal Neo
R
DNA
RNA
protein
Poly A TrapPoly A Trap
Endogenous gene X
lac ZSA
neo
SA lac Z neo
Nature Reviews Genet 2:756 (2001)Nature Reviews Genet 2:756 (2001)

15. Special types of TrappingSpecial types of Trapping
1. Secretory trap
2. Cre-loxP system
4. Protein trap
3.Chromosomal deletion using
Negative selection
(keeping functional objectives in view)

16. 1. Secretory Trap: Basic strategy1. Secretory Trap: Basic strategy

17. Secretory Trap: Improved strategySecretory Trap: Improved strategy
Protein

18. Coronal sections of
forebrains
showing PLAP
expression in
Secretory trap in
mouse at birth
Nature 410:174 (2001)Nature 410:174 (2001)
Trapped Gene name
Area of brain
Identification of role of specific gene during Identification of role of specific gene during
development development

19. 2. cre-loxP mediated excision2. cre-loxP mediated excision

20. Construct with loxP site:
loxPloxP
DNA after
integration
Exon from
endogenous gene
RNA

21. 3. Protein Trap3. Protein Trap
Brief Funct Genomic Proteomic. 2:137 (2003)
Introns

22. Expression of trapped genes (GFP) in Expression of trapped genes (GFP) in
different developmental stagesdifferent developmental stages
FEBS Letters 480:63 (2000)FEBS Letters 480:63 (2000)

23. Vector DeliveryVector Delivery
1. Chemical method:
using reagents to package vector DNA
2. Electroporation:
Applying electrical forces to
enhance cell membrane pores
3. Biological system: Viral infection
with adeno, lenti or retroviral vectors

24. Identification of insert locationIdentification of insert location
1.Using a Rescue Vector strategy
2.Using a Expression of the
reporter/marker gene- RACE

25. Applications of gene trapApplications of gene trap

26. Gene Trap
Identifying New Genes
Labeling Cell LineagesEffect of mutagenesis
Induced Deletions
Chromosome Trap

27. Gene trap helps in annotation of genome and Gene trap helps in annotation of genome and
identifying new genes with unknown functionidentifying new genes with unknown function
Nucleic Acids Research 32: 3995 (2004)Nucleic Acids Research 32: 3995 (2004)

28. Studying X- chromosome Inactivation in Human
Alleles
Gel
Nucleic Acids
Research (2004)
Allele 1 Allele 2
neo
ES cells Differentiated ES cells
No-inactivation
Non-random
Inactivation
Random
Inactivation
neo
selection
neo
Allele 1 Allele 2Allele 1 Allele 2
neo
50% 50%
Allele 1 Allele 2
neo
Allele 1 Allele 2
neo

29. Using gene trap method this study concluded thatUsing gene trap method this study concluded that::
In human extra-embryonic tissue (placenta),
X inactivation is of non- random type.
One can learn more about epigenetics
using trapped clones and identify
imprinted genes (those are expressed
from one chromosome only) .
Nat Genet. 28:310 (2001). Nat Genet. 28:310 (2001).

30. Should not be confused with Should not be confused with
gene targetinggene targeting

31. • Integration of genomic DNA into
mammalian cell genome by homologous
sequence recombination.
•It is usually used to create direct
mutagenesis in mammalian cell particularly
in mouse embryonic stem cell.
•Phenotypic consequence of specific genetic
modification can be assessed in the
organism (e.g. loss of function ).
What is gene targeting?What is gene targeting?

32. Gene TargetingGene Targeting
gene
neoTK
neo
x x
Negative selectionPositive selection
Chromosome
Vector
Targeted locus
Homologous recombination

33. Limitations of gene trapLimitations of gene trap
1. Lack of effective prescreening of trapped genes.
2. Integration of multiple copies of the trap vector etc.
3. Biasness of the trapping vectors.
4. Cannot be used for genes which are permanently
switched off.
5. Particular gene of interest may not be mutated.
6. Effect of Differential and Alternative Splicing.