14. In transgenic mice, the orientation an location of the loxP sites determine whether Cre recombinase induces a deletion, an inversion or a chromosomal translocation. If a researcher wants to put loxP sites in such a manner that only inversion will take place, which one of the following construct best justifies their intension. Gene X is the target gene.

Only construct (3), where gene X is flanked by two loxP sites in opposite orientation, will undergo inversion without deletion when Cre recombinase is expressed.

Concept: Cre‑loxP orientation and outcome

Cre recombinase recognizes 34‑bp loxP sites and catalyzes recombination between two loxP sequences. When loxP sites are in the same (direct repeat) orientation on one DNA molecule, Cre excises the intervening fragment as a circular piece, causing a deletion in the chromosome. When loxP sites are in opposite (inverted, head‑to‑head) orientation on the same molecule, recombination inverts the intervening DNA segment in situ, rather than deleting it.

Interpreting the question

The question asks which construct places loxP sites “in such a manner that only inversion will take place,” with gene X as the target gene. The correct design must therefore:

• Place two loxP sites on the same chromosome.

• Arrange them in inverted orientation around gene X so that Cre induces inversion of gene X, not deletion.

Option‑wise explanation

Option (1)

In this construct, gene X lies upstream, followed by two loxP sites that appear in the same direction (direct repeats) on the same side of gene X. Gene X is not actually flanked by the two loxP sites, so the recombination event occurs only between the two downstream loxP sites, excising the small fragment between them and leaving gene X untouched. This leads to deletion of the intervening DNA, not inversion of gene X, so option (1) does not satisfy the requirement.

Option (2)

Here, one loxP site is placed upstream of gene X and another downstream, but both arrows show the same orientation (direct repeat). Recombination between directly repeated loxP sites excises the DNA located between them, which in this design includes gene X; therefore, Cre activity would delete gene X from the chromosome. Because the outcome is deletion rather than inversion, option (2) is incorrect.

Option (3) – Correct

In this construct, gene X is positioned between two loxP sites that face each other (inverted, head‑to‑head orientation), clearly flanking the gene. Recombination between inverted loxP sites causes the DNA segment between them to flip 180° in place, producing an inversion of gene X while preserving its presence in the chromosome. Since the gene is bounded by oppositely oriented loxP sites and not duplicated elsewhere, only inversion occurs, making option (3) the correct answer.

Option (4)

In option (4), gene X is upstream, followed by two loxP sites that appear to be in opposite orientations but are both placed on the same side (downstream) of gene X rather than flanking it. In this situation gene X is outside the recombination cassette, so any Cre‑mediated event will involve only the segment between the two loxP sites, not gene X itself. Therefore, this construct does not invert gene X and cannot fulfill the experimental goal, so option (4) is also incorrect.

---

Introduction

The Cre‑loxP recombination system is a powerful tool in transgenic mice for generating precise genomic modifications such as deletions, inversions and chromosomal translocations. In exam questions on mouse genetics, understanding how the relative orientation and position of loxP sites dictates each outcome is essential for predicting whether a construct will delete or invert a target gene like gene X.

Core explanation for students

• When loxP sites are arranged as direct repeats flanking a DNA segment, Cre excises that segment and leaves behind a single loxP site, resulting in a stable deletion of the floxed gene.

• When loxP sites are positioned in inverted orientation around a segment on the same chromosome, Cre recombination flips that segment, causing a reversible genomic inversion that keeps the DNA in place.

• Therefore, to design a mouse in which Cre activity leads only to inversion of gene X and not its deletion, gene X must be precisely flanked by two loxP sites in opposite orientation, as depicted in construct (3).