Transcript GENE TRAPPING Paras Yadav*, Jaspreet Singh Arora*, Sachinandan De*, Tirtha Kumar Datta*, Surender Lal Goswami*, Aarti Bhardwaj$, Shalini Jain# and Hariom Yadav# *Animal Biotechnology,

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
What is gene trapping ?
Gene trapping is a form of insertional mutagenesis
specifically designed to disrupt gene function by
producing intragenic integration events.
Evans, M.J. (1998) Dev. Dyn., 212, 167-169
• 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.
Regulatory Components of a Gene –
important for its expression
Enhancer a set of short sequence elements which stimulate
transcription of a gene.
Promoter a combination of short sequence elements to which
RNA polymerase binds in order to initiate 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.
Basic Strategy in Gene Trap
Gene 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
Components of gene trap:
• Mouse or human embryonic stem cell (for
mammalian model)
• Entrapment vector construct having the
reporter gene and selectable marker.
Reporter 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
Selectable Markers
Positive selection:
• Neomycin phosphotransferase gene (neoR)
• Puromycin selection (Puro)
Negative selection:
• Herpes Simplex Thymidine kinase gene (hsv-tk)
• Diphtheria toxin gene
Types of vectors
•
•
•
•
Enhancer trap vector
Promoter trap vector
Gene trap
poly A trap
Enhancer Trap
Endogenous gene X
Vector
promoter
Enhancer
Exon 1
Exon 2
DNA
RNA
protein
proteinX
lac Z
neo
lac Z
pA
Exon 3
Vector Integration
promoter
P'
neo
lac Z
neo
β-gal
NeoR
pA
Promoter Trap
Endogenous gene X
P'
lac Z
neo
pA
Vector Integration
DNA
lac Z
RNA
protein
lac Z
proteinX
β-gal
neo
neo
NeoR
pA
Gene Trap
Endogenous gene X
P'
lac Z
SA
Vector Integration
SA
DNA
lac Z
neo
Spliced transcript
RNA
protein
lac Z
SA
proteinX
β-gal
neo
NeoR
neo
pA
pA
Poly A Trap
Endogenous gene X
P'
lac Z
SA
neo
SD
pA
Vector Integration
SA
DNA
lac Z
neo
pA
SA
RNA
lac Z
neo
pA
protein
proteinX
β-gal
NeoR
Nature Reviews Genet 2:756 (2001)
Special types of Trapping
(keeping functional objectives in view)
1. Secretory trap
2. Cre-loxP system
3.Chromosomal deletion using
Negative selection
4. Protein trap
1. Secretory Trap: Basic strategy
Secretory Trap: Improved strategy
Protein
Identification of role of specific gene during
development
Trapped Gene name
Coronal sections of
forebrains showing
PLAP expression in
Secretory trap in
mouse at birth
Area of brain
Nature 410:174 (2001)
2. cre-loxP mediated excision
Construct with loxP site:
loxP
DNA after
integration
RNA
Exon from
endogenous gene
3. Protein Trap
Introns
Brief Funct Genomic Proteomic. 2:137 (2003)
Expression of trapped genes (GFP) in
different developmental stages
FEBS Letters 480:63 (2000)
Vector 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
Identification of insert location
1.Using a Rescue Vector strategy
2.Using a Expression of the
reporter/marker gene- RACE
Applications of gene trap
Labeling Cell Lineages
Effect of mutagenesis
Gene Trap
Identifying New Genes
Chromosome Trap
Induced Deletions
Gene trap helps in annotation of genome and
identifying new genes with unknown function
Nucleic Acids Research 32: 3995 (2004)
Studying X- chromosome Inactivation in Human
ES cells
Differentiated ES cells
No-inactivation
Non-random
Inactivation
Random
Inactivation
neo
neo
neo
Alle le 1
Alle le 2
Alle le 1
Alle le 2
neo
Alle le 1
Alle le 2
neo
Alle le 1
50%
Alle le 2
selection
neo
Alle le 1
Alle le 2
50%
Alleles
Gel
Nucleic Acids
Research (2004)
Using 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).
Should not be confused with gene
targeting
What is gene targeting?
• 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 ).
Gene Targeting
Negative selection
Positive selection
TK
neo
x
Vector
x
gene
Chromosome
Homologous recombination
neo
Targeted locus
Limitations 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.