15. A student was asked to design a knockout cassette for specifically deleting the p53 gene from the prostate gland of mice. Which one of the following pairs of cassettes will ensure deletion of the gene?
The correct pair of cassettes is option (3), where Cre recombinase is expressed under a tissue‑specific promoter in the prostate and the p53 gene is flanked by loxP sites (floxed) on both sides so that Cre can excise it only in that tissue.
Introduction
Designing a tissue‑specific gene knockout requires combining a tissue‑specific promoter driving Cre recombinase with a target gene that is flanked by loxP sites, so that deletion occurs only where Cre is expressed. In this CSIR NET life sciences question, the task is to choose the correct pair of cassettes that will specifically delete the p53 gene in the prostate gland of mice, using a tissue‑specific promoter and loxP‑flanked p53.
Concept: Cre‑LoxP tissue‑specific knockout
In the Cre‑LoxP system, Cre recombinase recognizes two loxP sites oriented in the same direction and excises the DNA segment between them, leaving a single loxP behind. When Cre is placed under a tissue‑specific promoter (TSP), Cre is produced only in that tissue, so recombination and deletion occur only in that organ while the rest of the body retains a functional gene. For a conditional knockout, the endogenous gene (here p53) must be “floxed” by inserting loxP sites on both sides of essential exons.
Option (1) analysis
In option (1), the left cassette uses a tissue‑specific promoter (TSP) driving Cre recombinase, which is correct for restricting Cre expression to the prostate. However, in the right cassette, the p53 gene has only a single loxP site drawn at one end of the gene. With only one loxP site present, Cre has nothing to recombine with and cannot excise any DNA segment, so the p53 gene will not be deleted in the prostate or anywhere else. Therefore, option (1) does not ensure deletion of the p53 gene.
Option (2) analysis
In option (2), the cassette on the left has Cre placed after the promoter region of the p53 gene rather than under a separate tissue‑specific promoter. This configuration would make Cre expression driven by the p53 gene’s own promoter, and it is not prostate specific. Consequently, Cre could be expressed in any tissue where p53 is active, losing the required organ specificity. In addition, the second cassette shows the p53 gene without properly positioned loxP sites on both sides, so even if Cre is expressed, it will not correctly delete p53 in a tissue‑restricted manner. Hence, option (2) is not a valid strategy.
Option (3) analysis (correct)
In option (3), the first cassette contains a tissue‑specific promoter (TSP) driving Cre recombinase, so Cre is synthesized only in the prostate gland cells. The second cassette shows the endogenous p53 gene with two loxP sites flanking the critical coding region (one loxP on each side of p53), i.e., a “floxed” p53 allele. When these two mouse lines are crossed, the progeny that carry both cassettes will express Cre in the prostate, where Cre will recognize the two loxP sites surrounding p53 and excise the intervening DNA, knocking out p53 specifically in prostate tissue while keeping it intact in all other tissues. Therefore, option (3) correctly ensures prostate‑specific deletion of the p53 gene.
Option (4) analysis
In option (4), the left cassette again correctly shows a tissue‑specific promoter driving Cre. However, the p53 cassette on the right has the loxP sites placed outside the gene region or in an incorrect orientation such that the functional coding sequence is not properly floxed. If loxP sites do not flank the essential exons or are not both in the same direction, Cre will either fail to excise the gene or produce an unintended rearrangement rather than a clean deletion. Due to this improper floxing of p53, option (4) does not reliably delete the p53 gene specifically in the prostate.
