Pronuclear microinjection — at what time relative to pronuclear fusion is foreign DNA optimally microinjected into a fertilized mammalian egg?

Introduction / Context:
Classical production of transgenic animals often uses pronuclear microinjection, in which DNA is delivered into one pronucleus (commonly the male) of a zygote prior to syngamy. Successful timing increases the chance of genomic integration and germline transmission.

Given Data / Assumptions:

• The fertilized egg presents two visible pronuclei before fusion.
• Microinjection targets a pronucleus, not the metaphase plate after fusion.
• Goal: stable genomic integration and development.

Concept / Approach:

Before pronuclear fusion, the nuclear membranes and chromatin state facilitate DNA deposition and subsequent integration during the first DNA replication. Injecting too late (post-fusion) reduces efficiency and may increase mosaicism.

Step-by-Step Solution:

Verification / Alternative check:

Standard rodent transgenesis protocols time injections to the pronuclear stage, confirmed by visible pronuclei under microscopy.

Why Other Options Are Wrong:

(a) and (e) happen too late, increasing mosaicism; (c) is impractical to time precisely; (d) timing clearly affects outcome and is not arbitrary.

Common Pitfalls:

Injecting into the cytoplasm instead of a pronucleus; delaying injection until after pronuclear fusion; causing excessive mechanical damage.

Final Answer:

Before the fusion of male and female nuclei (pronuclear stage)