17. In order to create an animal whose prostate gland is devoid of the p53 gene, engineered animals were created by cloning specially designed knock-out cassettes. Identify the most appropriate cassette combination for creating the knock out animal by breeding.

The correct cassette combination is: Mouse 1: (1) MMTV–Cre × Mouse 2: (3) GOI–GFP–GSP (loxP-flanked p53), because this pairing ensures Cre is expressed specifically in the prostate and excises the p53 gene only in that tissue, creating a prostate‑specific p53 knockout animal.

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Introduction

Conditional gene targeting using the Cre‑Lox system is widely used to create tissue‑specific knockouts, such as a prostate specific p53 knockout mouse for cancer studies. In this CSIR NET‑style question, different transgenic mouse lines carry cassettes for MMTV‑Cre and floxed p53 (gene of interest, GOI) with GFP and tissue‑specific promoters, and the task is to select the correct breeding combination to remove p53 only in the prostate gland. Selecting the right design requires understanding how Cre expression, loxP‑flanked alleles, GFP reporters and tissue‑specific promoters interact in vivo.

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Background: Cre‑Lox and cassette design

• Cre recombinase recognizes loxP sites flanking a DNA segment (the floxed region) and excises it, inactivating the gene of interest in cells where Cre is expressed.

• Tissue‑specific promoters such as probasin or other prostate‑specific promoters restrict Cre activity to the prostate epithelium, enabling conditional p53 loss only in that tissue.

• Reporter cassettes with GFP are often placed so that GFP is expressed only after Cre‑mediated recombination, confirming successful knockout in the target tissue.

In this question, “MMTV” is a promoter cassette driving Cre (originally from mouse mammary tumor virus but here acting as a tissue‑specific promoter in the gland of interest), GOI is the p53 gene, GSP is a gene‑specific (prostate) promoter, and GFP is a reporter.

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Option‑by‑option analysis

The options pair MICE 1 (Cre‑expressing lines) with MICE 2 (floxed p53/GOI reporter lines). The goal is:

• Cre must be expressed only in the prostate, not systemically.

• The p53 (GOI) cassette must be flanked by loxP sites and arranged so that Cre removes or functionally inactivates it in prostate cells.

• An ideal design can additionally use GFP to report successful recombination, but GFP is not mandatory for correctness.

Option 1: MICE1 (1) MMTV–Cre × MICE2 (1) GFP–GOI–GFP

• MICE1 (1) carries MMTV–Cre, so Cre is expressed under a tissue‑specific promoter; this part is acceptable.

• MICE2 (1) shows GFP–GOI–GFP but, as typically drawn in such exam questions, the GOI is not clearly flanked by loxP sites; the two GFP cassettes are on either side but there is no indication that Cre cutting will delete p53 rather than only rearranging GFP.

• Without explicit loxP‑flanking of the GOI, this construct is ambiguous and does not guarantee functional knockout of p53 while leaving the rest of the locus intact.

Conclusion: Option 1 is not the best choice because the floxed structure of p53 is not clearly defined.

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Option 2: MICE1 (2) MMTV–Cre (modified) × MICE2 (2) GFP–GOI–GFP (different orientation)

• MICE1 (2) still uses MMTV–Cre, thus tissue‑specific expression of Cre remains acceptable.

• MICE2 (2) again arranges GFP–GOI–GFP, but here one GFP is oriented oppositely (shown by the arrow in the original figure), suggesting a reporter‑switch design where Cre might invert or activate GFP rather than excise the GOI.

• Such designs are generally used for lineage tracing or conditional activation of reporters, not for a clean deletion of a tumor‑suppressor gene like p53.

Conclusion: Option 2 primarily manipulates GFP expression and does not reliably ensure complete p53 deletion in prostate cells.

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Option 3: MICE1 (3) MMTV–Cre × MICE2 (3) GOI–GFP–GSP (loxP‑flanked p53)

• MICE1 (3) expresses Cre under the MMTV (here, prostate‑specific) promoter, which restricts recombinase activity to the prostate gland, satisfying the tissue‑specific expression requirement.

• MICE2 (3) contains a cassette where the GOI (p53) is positioned so that it is flanked by loxP sites (indicated by the two arrowheads in the exam diagram) with GFP and a gene‑specific promoter (GSP) arranged to report or drive expression after deletion. In standard floxed designs, Cre excises the GOI between loxP sites, and the downstream GFP comes under GSP control only in recombined cells.

• Crossing these two lines yields offspring in which Cre is produced only in prostate cells, excising the floxed p53 there and leaving p53 intact in all other tissues. This matches the requirement: “prostate gland is devoid of p53 gene, while the rest of the animal retains p53.”

Conclusion: Option 3 provides a correct conditional p53 knockout specific to the prostate, and is therefore the correct answer.

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Option 4: MICE1 (4) MMTV–Cre × MICE2 (4) GFP–GOI–GFP (no tissue‑specific promoter for GOI)

• MICE1 (4) again provides MMTV–Cre, so Cre is prostate‑restricted.

• MICE2 (4) has GFP–GOI–GFP but lacks the GSP (prostate‑specific promoter) element that ensures proper spatial control and expression before and after recombination; this construct risks mis‑expression or incomplete inactivation of p53 and does not provide clear recombination‑dependent GFP readout.

• Because the GOI is not clearly floxed with a defined downstream prostate promoter, Cre activity may not efficiently remove p53 or may disrupt additional sequences, making this an inferior design for a clean, prostate‑restricted knockout.

Conclusion: Option 4 fails to provide a robust, specifically regulated p53 knockout cassette and is not the best answer.

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Summary table: Why option 3 is correct

The best cassette combination to create an animal whose prostate gland lacks p53, while other tissues retain normal p53 expression, is therefore Option 3: MICE1 (3) × MICE2 (3).