20. Construction of knockout mice may be performed using the Cre-LoxP system. Eventually, the Cre recombinase of the bacteriophage P1 mediates site- specific recombination at a 34 bp sequence, lox P. From the following statements, choose the INCORRECT event:
(1) The alteration of the chromosomal copy of the target gene requires a guide RNA.
(2) The loxP containing mice should not express Cre recombinase prior to mating.
(3) The Cre recombinase can be expressed by an inducible promoter.
(4) Induction of the promoter results in the expression of Cre, recombination at lox P sites and excision of the sequence in between.
Introduction
The Cre-LoxP system enables precise construction of knockout mice by using Cre recombinase from bacteriophage P1 to mediate site-specific recombination at 34 bp loxP sequences, allowing conditional gene excision flanked by loxP sites (floxed alleles). This technique is vital for studying gene functions in specific tissues or developmental stages without global knockouts. In CSIR NET Life Sciences exams, questions test understanding of its mechanisms versus modern tools like CRISPR.
Option Analysis
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Option (1): The alteration of the chromosomal copy of the target gene requires a guide RNA.
This is incorrect. Traditional Cre-LoxP relies on homologous recombination to insert loxP sites into the target gene, with no guide RNA needed—Cre directly recognizes loxP for recombination. Guide RNA is specific to CRISPR-Cas9 systems, sometimes combined with Cre-LoxP for efficiency, but not required in standard Cre-LoxP knockout mice construction. -
Option (2): The loxP containing mice should not express Cre recombinase prior to mating.
This is correct. Floxed (loxP-containing) mice must lack Cre expression before mating with Cre-driver lines to prevent premature recombination and unwanted early gene excision, ensuring controlled conditional knockouts. -
Option (3): The Cre recombinase can be expressed by an inducible promoter.
This is correct. Inducible promoters (e.g., tamoxifen-responsive) allow temporal control of Cre expression, enabling recombination at desired times post-mating for precise studies. -
Option (4): Induction of the promoter results in the expression of Cre, recombination at loxP sites and excision of the sequence in between.
This is correct. Promoter induction triggers Cre expression, which binds loxP sites in the same orientation, excising the intervening floxed sequence as a circle, achieving tissue- or time-specific knockout.
Correct Answer and Exam Insight
The incorrect event is option (1), as guide RNA is absent in pure Cre-LoxP methodology, distinguishing it from CRISPR hybrids.
