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 Enhancera set of short sequence elements which stimulate transcription of a gene. Promotera combination of short sequence elements to which RNA polymerase binds in order to initiate transcription of a gene. Polyadenylationaddition of typically 200 A residues to the 3' end of a mRNA. The poly(A) tail is important for stabilizing mRNA.
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.colilacZ 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 P' promoter neo lac Z pA pA Exon 1 Exon 2 Exon 3 Enhancer Vector Integration DNA neo promoter lac Z lac Z neo RNA proteinX β-gal NeoR protein
Promoter Trap Endogenous gene X P' lac Z neo pA pA Vector Integration DNA lac Z neo RNA neo lac Z proteinX protein β-gal NeoR
Gene Trap Endogenous gene X P' lac Z neo SA pA pA Vector Integration lac Z SA neo DNA Spliced transcript neo RNA lac Z SA proteinX protein NeoR β-gal
Poly A Trap Endogenous gene X P' lac Z neo SA SD pA Vector Integration neo SA lac Z DNA pA SA lac Z neo RNA pA proteinX protein NeoR β-gal 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
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)
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
Labeling Cell Lineages Effect of mutagenesis Gene Trap Chromosome Trap Identifying New Genes 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 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).
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 Vector TK neo x x Chromosome gene Homologous recombination Targeted locus neo
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.