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) Dev. Dyn., 212, 167-169 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 – important for its expression

5. Basic Strategy in Gene Trap

6. 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

7. 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 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 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 vectors Enhancer trap vector Promoter trap vector Gene trap poly A trap

11. promoter Enhancer DNA RNA protein proteinX proteinX β-galNeo R promoter lac Z P' pA Endogenous gene X Vector Integration Vector neo lac Z neo Enhancer Trap lac Zneo Exon 1Exon 2Exon 3

12. Endogenous gene X lac Z pA P' neo pA Vector Integration DNA RNA protein proteinX proteinX β-galNeo R lac Z neo Promoter Trap lac Z neo

13. SA Endogenous gene X SA lac Z pA P' neo pA Vector Integration DNA RNA protein Spliced transcript proteinX proteinX β-gal Neo R Gene Trap lac Z neo SA lac Z neo

14. SA pA P' neo SD lac Z Vector Integration pA proteinX proteinX β-gal Neo R DNA RNA protein Poly A Trap Endogenous gene X lac ZSA neo SAlac Zneo Nature Reviews Genet 2:756 (2001)

15. Special 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 strategy

17. Secretory Trap: Improved strategy Protein

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

19. 2. cre-loxP mediated excision

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

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

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

23. 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

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

25. Applications of gene trap

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

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

28. Studying X- chromosome Inactivation in Human Alleles Gel Nucleic Acids Research (2004)

29. 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).

30. Should not be confused with gene 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?

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

33. 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.