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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 P' neo pA Vector Integration pA

lac Z

Enhancer

Exon 1

Exon 2

Exon 3

DNA promoter lac Z neo

RNA

lac Z

neo

protein

proteinX

-gal

NeoR

Promoter Trap
Endogenous gene X P' lac Z pA Vector Integration neo pA

DNA

lac Z

neo

RNA

lac Z

neo

protein

proteinX

-gal

NeoR

Gene Trap
Endogenous gene X P' lac Z SA Vector Integration pA neo pA

DNA Spliced transcript RNA

SA

lac Z

neo

SA

lac Z

neo

protein

proteinX

-gal

NeoR

Poly A Trap
Endogenous gene X P' lac Z SA Vector Integration pA neo SD

DNA

SA

lac Z

neo pA

RNA

SA

lac Z

neo pA

protein

proteinX

-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

Exon from endogenous gene

DNA after integration RNA

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 neo neo

selection
Allele 1

neo

Allele 1

Allele 2

Allele 1

Allele 2

Allele 1

Allele 2

Allele 1 50%

Allele 2 50%

Allele 2

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 TK Positive selection neo

Vector

x
gene
Homologous recombination neo

Chromosome

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.

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