#Cre recombinase

Recombination

Cre recombinase belongs to which family?

Site-specific recombinases are divided into serine and tyrosine families based on nucleophilic residue forming covalent protein-DNA intermediate during strand exchange. Cre recombinase belongs to tyrosine family alongside lambda integrase, Flp, and XerC/D. Catalytic tyrosine at C-terminus attacks phosphodiester bond generating 3'-phosphotyrosine linkage and free 5'-hydroxyl, proceeding through sequential single-strand exchanges forming Holliday junction intermediate. Tetramer synchronizes two rounds of cleavage and ligation without ATP or external energy, using binding energy and base pairing. Conserved Arg-His-Arg triad assists active site chemistry and transition state stabilization during recombination.

Ref: NCBI Bookshelf, Molecular Biology, Tyrosine Recombinase Family and Cre Catalytic Mechanism

View Details

Gene Tar getting

A researcher sees Cre-mediated excision only in neurons. What was used?

Tissue specific conditional knockout exploits Cre expression under cell type specific promoter. Floxed target gene remains intact in all cells. Cre recombinase driven by neuron specific enolase or Synapsin-1 promoter is transcribed only in neurons, so loxP flanked exon excision occurs exclusively in neuronal lineage, leaving other tissues wild type. This spatial control avoids lethality of global deletion. Inducible systems add temporal regulation, but neuron restricted pattern directly indicates use of neuron specific regulatory element controlling Cre recombinase expression cassette.

Ref: NCERT Biology Class XII Principles on Klenow fill-in labeling, Lehninger Chapter 9 DNA cloning techniques, and Molecular Cloning by Sambrook Chapter 10 documenting end-labeling of cohesive termini.

View Details

Gene Tar getting

What would result from placing loxP sites flanking exon 2 of a gene in same orientation?

Placing loxP sites in same orientation flanking exon 2 and intronic regions preserves gene function until Cre acts. Cre recombinase synapses two directly repeated loxP sites, forming a loop and catalyzing strand exchange. Recombination excises the looped DNA as circular molecule containing exon 2 and one loxP site, leaving single loxP scar in chromosome. Loss of coding exon causes frameshift mediated nonsense or deletion of essential domain, achieving conditional knockout after tissue specific Cre activation without affecting other tissues developmentally.

Ref: NCERT Biology Class XII Principles on Klenow fill-in labeling, Lehninger Chapter 9 DNA cloning techniques, and Molecular Cloning by Sambrook Chapter 10 documenting end-labeling of cohesive termini.

View Details

Gene Tar getting

In conditional knockout using Cre, gene excision happens when:

Conditional knockout overcomes embryonic lethality by floxing essential exons with loxP sites placed in same orientation flanking critical region intronically, preserving normal splicing without Cre. Excision occurs only upon Cre expression which recombines directly repeated loxP sites, deleting intervening exon and creating frameshift or null allele. Tissue specific or tamoxifen inducible Cre drivers then restrict deletion temporally and spatially. Requirement of same direction loxP ensures deletion rather than inversion, producing irreversible loss of function in selected cells only in vivo efficiently.

Ref: NCERT Biology Class XII Principles on Klenow fill-in labeling, Lehninger Chapter 9 DNA cloning techniques, and Molecular Cloning by Sambrook Chapter 10 documenting end-labeling of cohesive termini.

View Details

Gene Tar getting

The Cre recombinase enzyme recognizes which DNA element?

Cre is a 38 kDa tyrosine recombinase encoded by bacteriophage P1 that catalyzes site-specific recombination without cofactors. Its target is loxP, a 34 base pair sequence containing an asymmetric 8 base pair core spacer flanked by two 13 base pair inverted repeats where Cre binds as dimer. Recombination between two loxP sites excises or inverts intervening DNA depending on orientation. Specificity for loxP distinguishes Cre from Flp which recognizes FRT, making Cre-loxP a powerful conditional genome manipulation tool in mammals.

Ref: NCERT Biology Class XII Principles on Klenow fill-in labeling, Lehninger Chapter 9 DNA cloning techniques, and Molecular Cloning by Sambrook Chapter 10 documenting end-labeling of cohesive termini.

View Details