Functional assay for identification of loss-of-function mutations in genes

A method for classifying relative deleteriousness of a mutation in a nucleic acid sequence of a gene to a mammalian cell comprising:

• providing an isolated mammalian cell comprising a genome comprising a single functional copy of a first gene and a non-functional copy of the first gene;
• transforming the cell with a nucleic acid construct comprising a nucleic acid sequence from a second gene comprising the mutation to be classified, wherein the nucleic acid sequence from the second gene comprises a mutated first gene or a mutated fragment of the first gene, wherein the nucleic acid sequence from the second gene is flanked by two nucleic acid sequences to allow recombination with the genome of the cell in the first gene;
• detecting a frequency of recombination of the nucleic acid sequence from the second gene in the functional copy of the first gene,
• detecting a frequency of recombination of the nucleic acid sequence from the second gene in the non-functional copy of the first gene,
• comparing the frequency of recombination in the functional copy of the first gene to the frequency of recombination in the non-functional copy of the first gene, wherein an equal or higher frequency of recombination of the nucleic acid sequence from the second gene in the functional copy of the first gene compared to the non-functional copy of the first gene is indicative of a nondeleterious mutation to a mammalian cell, and wherein a lower frequency of recombination of the nucleic acid sequence from the second gene in the functional copy of the first gene compared to the non-functional copy of the first gene is indicative of a deleterious mutation to a mammalian cell.