Table 2.

Validation of genetic alterations

Allele production	Validation criteria	Assay type
Classical gene targeting	Targeting the correct locus	Southern blot, loss-of-allele qPCR/ddPCR and/or long-range PCR
	Structure of the targeting cassette	PCR and sequencing of targeting cassettes
	Functionality of targeting cassette	PCR and sequencing of key functional sequences, functional assays
	Absence of extra copies	Southern blot, qPCR/ddPCR, plasmid backbone PCR
Additive transgenesis	Transgene	qPCR/ddPCR, Southern blot
		qPCR/ddPCR or expression study in subsequent generations
	Locus of insertion	Fluorescence in situ hybridisation, inverse PCR, targeted locus amplification, X-drop analysis
Genome-editing	Sequence of the region of interest	PCR and Sanger or next-generation sequencing
	Copy number of mobilised segments and donor sequences	ddPCR, qPCR, Southern blot