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#loxP sites

6 public questions tagged with this topic.

Recombination

loxP sites in inverted orientation lead to

Outcome control by loxP orientation underpins Cre utility. Recombination between directly repeated loxP excises intervening segment as circle, often desirable for deletion. When repeats inverted, circular product cannot form because joining opposite strands flips orientation instead of deleting. After first strand exchange forming Holliday junction, resolution in inverted geometry rotates segment 180 degrees, reversing direction of coding sequences between sites. This inversion reaction is reversible as both resultant loxP sites remain in chromosome, allowing conditional invertible modules for

Ref: Lodish et al., Molecular Cell Biology, 9th ed., Chapter 8: Inverted loxP Sites Lead to Inversion

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Recombination

Orientation of loxP sites in inverted direction leads to

When two loxP sites reside on same DNA molecule in inverted orientation, Cre recombinase still forms tetrameric synapse but alignment is antiparallel, causing exchange to flip intervening segment rather than delete it. Cleavage in spacer region and rejoining in opposite direction inverts DNA between sites, retaining both loxP sites on chromosome with reversed orientation of internal sequence. This principle underlies generation of invertible gene traps, switching cassettes, and orientation-specific lineage tracing. Inversion is reversible because sites remain, allowing repeated flipping if Cre

Ref: Lodish et al., Molecular Cell Biology, 9th ed., Chapter 8: Inversion Outcome of Inverted loxP Sites

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Recombination

Orientation of loxP sites in same direction leads to

Outcome of Cre-loxP recombination is dictated by relative orientation and location of loxP sites. When two loxP sites flank a DNA segment on same chromosome in direct repeat orientation, Cre-mediated synapsis brings sites together in parallel alignment, reciprocal exchange excises intervening DNA as circular product bearing one loxP and leaves single loxP at chromosomal locus. This deletion reaction is widely used for conditional knockouts using floxed alleles. Excision is essentially irreversible in absence of reintroduction, especially when circular product lacks replication origin and is lo

Ref: Alberts et al., Molecular Biology of the Cell, 7th ed., Chapter 8: Cre-loxP Deletion by Directly Repeated Sites

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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 g

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.

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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.

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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

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.

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