13. Which one of the following statements about Cre- mediated site-specific recombination at loxP sites is INCORRECT?
(1) When loxP sites flanking a test sequence are oriented in same direction, Cre mediates the excision of the intervening sequence
(2) LoxP sites in inverted orientation around an intervening sequence lead to inversion upon action by Cre recombinase
(3) LoxP sites recognized by the Cre recombinase ar palindromic around a spacer sequence
(4) LoxP-Cre system cannot be used to generate translocation between chromosomes.

Cre-mediated site-specific recombination at loxP sites is a well-established genetic tool that allows precise DNA rearrangements depending on the orientation of loxP sites. The system operates such that when loxP sites flank a DNA sequence in the same direction, Cre recombinase excises the intervening sequence, whereas if the loxP sites are in inverted orientation, the intervening sequence is inverted. The loxP site consists of palindromic 13 base-pair sequences flanking an 8 base-pair asymmetric spacer, making the site directional. Contrary to one of the statements in the question, the Cre-loxP system can be used to generate chromosomal translocations between chromosomes, which has been demonstrated in experimental mouse models.

Detailed explanation of the options:

1. Correct: When loxP sites flank a sequence in the same direction, Cre recombinase catalyzes excision of the intervening DNA as a circular segment, resulting in deletion of the sequence between them.

2. Correct: If loxP sites are in inverted orientation around a sequence, Cre catalyzes inversion of the intervening sequence rather than deletion.

3. Incorrect: The loxP sites are palindromic in their 13 bp regions, but the core 8 bp spacer sequence is asymmetric (non-palindromic), which imparts directionality to the loxP site. Thus, saying loxP sites are palindromic around a spacer sequence is misleading; the spacer itself is not palindromic.

4. Incorrect and hence the “INCORRECT” option in the question: The Cre-loxP system can indeed be used to generate chromosomal translocations between chromosomes, demonstrated in mouse embryonic stem cells and primary cells, making option (4) wrong as presented.

Hence, the incorrect statement is option (3) due to the inaccuracy regarding the palindromic nature of the loxP sites around the spacer sequence.

Introduction:
The Cre-LoxP recombination system is a powerful genetic engineering tool derived from bacteriophage P1. It enables precise DNA modifications such as excision, inversion, and translocation by recognizing specific loxP sites on DNA. The outcome of Cre recombinase activity depends largely on the orientation of these 34-base pair loxP sites. This article delves into the mechanism of Cre-mediated recombination, the significance of loxP site orientation, and dispels common misconceptions about its applications in chromosome engineering.

Detailed Explanation of Cre-loxP Recombination

• Cre recombinase recognizes 34 bp loxP sites consisting of two 13 bp palindromic sequences flanking an 8 bp asymmetric spacer, giving the site directionality.

• When two loxP sites flank a DNA segment in the same orientation, Cre excises the intervening DNA as a circular piece, effectively deleting that sequence.

• In the inverted orientation, Cre catalyzes inversion of the sequence between the loxP sites without excision.

• The system can also induce chromosomal translocations between non-homologous chromosomes, making it a versatile tool in genome engineering.

Explanation of Each Statement

• (1) Correct: Same direction loxP sites lead to excision.

• (2) Correct: Inverted loxP sites lead to inversion.

• (3) Incorrect: The loxP sites are palindromic except for the asymmetric 8 bp spacer, which is not palindromic.

• (4) Incorrect as stated: The system can be used to generate chromosomal translocations.

By understanding these principles, researchers can effectively manipulate genomes for functional studies and disease modeling.

This explanation clarifies the molecular mechanism and corrects common misunderstandings about Cre-loxP recombination.

References: The information is adapted from detailed scientific literature and reviews on Cre-loxP recombination and loxP site structure.​