The correct answer is: (4) Southern blot analysis.

Option-wise explanation

• (1) Male pronuclear insertion

  • This is a method of introducing the transgene (microinjection into the male pronucleus of the zygote).

  • It does not itself check whether the integrated transgene is intact or how many copies are present.

• (2) Fusion of enucleated egg with somatic cells

  • This refers to somatic cell nuclear transfer (cloning), another way to create genetically modified animals.

  • Again, it is a way of introducing genetic material, not of assessing transgene integrity.

• (3) Transfer into competent embryogenic cell

  • This describes introducing DNA into embryonic stem/embryogenic cells (e.g., in ES-cell–based transgenesis).

  • It does not analyze whether the final integrated transgene is full-length and correctly arranged.

• (4) Southern blot analysis – correct

  • Southern blotting uses restriction digestion of genomic DNA and hybridization with a transgene-specific probe.

  • It allows you to check:

    • Whether the transgene is present.

    • Its copy number (number of bands).

    • Whether it is full length or truncated and if there are major rearrangements (band sizes and patterns).

  • Therefore, it is the appropriate technique to validate integrity of the introduced transgene in the mouse genome.