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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
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.
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.
Selecting the clones with markers Identification of location of the insert in the clone Studying biological questions: Production of chimeras
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
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
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
Protein
Coronal sections of forebrains showing PLAP expression in Secretory trap in mouse at birth
Area of brain
3. Protein Trap
Introns
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
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
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
Gene Targeting
Negative selection TK Positive selection neo
Vector
x
gene
Homologous recombination neo
Chromosome
Targeted locus