Question

If I tried to knock in a gene for example KANMX and want to swap it with say some gene x, but, since, there can be double strand break in the DNA and KANMX may get inserted within the ds break instead of undergoing homologous recombination with gene x, in that case how can we identify the colonies which can be a mixture of the following :-

A.) Undergoing proper recombination and now having KANMX but not gene x
B.) Having both gene X and KANMX (how can we specifically identify these?)
C.) Undergoing no homologous recombination (one way is using G418 sulphate)

Answer 1

To identify colonies with different outcomes in the case of double-strand break repair, you can use a reporter gene for proper recombination, a different antibiotic resistance gene for colonies with both genes, and G418 sulfate for no homologous recombination.

Step-by-step explanation:

In the scenario where the KANMX gene gets inserted within the double-strand break instead of undergoing homologous recombination with gene X, there are several ways to identify the colonies with different outcomes:

1. To identify colonies that have undergone proper recombination but do not have gene X, you can use a reporter gene like the lacZ gene encoding beta-galactosidase. When gene X is properly recombined, it will disrupt the lacZ gene, resulting in white colonies on X-gal-containing media.
2. To identify colonies that have both gene X and KANMX, you can use a different antibiotic resistance gene like streptomycin. Inserting the cDNA into a plasmid with a restriction enzyme site within the gene will disrupt and inactivate it. Transformants containing recombinant plasmids can then be identified by replica plating and growth on streptomycin-containing media.
3. To identify colonies that have undergone no homologous recombination, you can use G418 sulfate, an antibiotic that inhibits the growth of cells without KANMX. The colonies that continue to grow in the presence of G418 sulfate have not undergone homologous recombination.