#Cre-lox system

Gene Tar getting

What is essential for inversion to happen in Cre-lox system?

In Cre-loxP system Cre binds 13 base pair inverted repeats surrounding 8 base pair asymmetric core of loxP. For inversion versus deletion decision, relative direction of core determines outcome. When core sequences point towards each other on same chromosome, meaning loxP sites in opposite orientation, Cre catalyzed recombination reverses intervening segment without loss. Direct repeats cause deletion. Thus requirement for inversion is antiparallel loxP orientation ensuring DNA strand exchanged leads to reversal rather than circular excision, employed in FLEX switches for reversible gene control.

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

Cre-FLEX system allows for:

Engineered Cre dependent vectors utilize double floxed inverted orientation or FLEX switch. Wild type and mutant loxP pairs are arranged antiparallel flanking gene in inverted orientation. In absence of Cre, transcription proceeds in wrong orientation or cassette blocked. Upon Cre expression, sequential recombination events invert coding sequence into correct orientation then excise two lox sites locking it permanently active or inactive. This enables irreversible flipping. Such reversible flipping before locking allows tight Cre dependent control for optogenetics, chemogenetics and cell type specific gene expression manipulation.

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

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

Which element is not necessary for gene targeting via homologous recombination?

Homologous recombination guided gene targeting requires extensive homology arms matching endogenous locus to direct strand exchange, and selectable marker like neo to identify integration events. Endogenous recombination machinery of embryonic stem cells including Rad51 mediated repair executes crossover. Origin of replication is plasmid bacterial element needed for propagation in E coli during vector construction and preparation but dispensable inside mammalian genome since integration occurs via chromosomal recombination, not autonomous replication. Therefore Ori is not required for actual gene targeting event in eukaryotic nucleus.

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 ‘floxed’ gene refers to a gene:

Floxed allele describes engineered genomic locus where essential region is flanked by loxP sequences, derived from phrase flanked by loxP. Construction inserts 34 base pair loxP sites into introns bracketing target exon via homologous recombination in embryonic stem cells, without disrupting transcription. Without Cre, floxed gene expresses normally. Upon crossing to Cre driver, recombination deletes floxed segment in desired tissue. Floxed terminology distinguishes conditional ready allele from constitutive knockout, enabling temporal control, lineage tracing and rescue experiments in mouse genetics.

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