You are making a knockout mouse, after neomycin (positive) selection you treat t

Question

You are making a knockout mouse, after neomycin (positive) selection you treat the surviving cells with gancyclovir (negative selection) and more cells die. What does cell death after gancyclovir treatment indicate?

1.     The targeting vector randomly inserted into the genome

The targeting vector did not insert into the genome

The targeting vector inserted into the genome by homologous recombination

1.     The targeting vector randomly inserted into the genome

The targeting vector did not insert into the genome

The targeting vector inserted into the genome by homologous recombination

Explanation / Answer

Generally, in producing a knockout mice selection markers are introduced into the homology region. A positive and a negative selection marker is used. In the question the positive marker is neomycin that confers G418 resistance. This ensures if the transformation ever happened. If cells survived after neomycin treatment it indicates that the transformation took place. The negative selection marker used is herpes simplex virus thymidine kinase (HSVtk) that makes the cells sensitive to gancyclovir treatment. This marker is situated outside the homology region. HSVtk selects for homologous recombination because it randomly integrates into the genome.

In the question cells survived after neomycin treatment which indicates that integration took place but after gancyclovir cells die which would mean that the vector randomly inserted into the genome. This would be the reason for cell death after gancyclovir treatment.

ANSWER: the targeting vector randomly inserted into the genome

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