Universal Southern blot protocol with cold or radioactive probes for the validation of alleles obtained by homologous recombination

Abstract

The widespread availability of recombineered vectors and gene targeted embryonic stem cells from large-scale repositories facilitates the generation of mouse models for functional genetic studies. Southern blotting validates the structure of these targeted alleles produced by homologous recombination, as well as indicating any additional integrations of the vector into the genome. Traditionally this technique employs radioactively-labelled probes; however, there are many laboratories that are restricted in their use of radioactivity. Here, we present a widely applicable protocol for Southern blot analysis using cold probes and alternative procedures employing radioactive probes. Furthermore, the probes are designed to recognise standardised regions of gene-targeting cassettes and so represent universally applicable reagents for assessing allelic integrity.

Keywords: Homologous recombination; Mouse; Southern blot; Targeting; Validation.

Fig. 1.

Design of Southern assay. The figure details a generic tm1a construct from the KOMP repository annotated with the positions of both the lacZ and neo probes used for Southern blot analysis. Four examples of restriction digests are annotated, which can be used to interrogate whether the cassette has integrated correctly on target into the genome; restriction digests using enzymes 1 and 2 are used to confirm correct insertion of the 5′ homology arm (HA) on target and restriction digests using enzymes 3 and 4 are used to verify the integrity of the 3′ HA. The expected sizes of the fragments of a correctly targeted allele and the probe(s) that can be used with each restriction digest are described in the table.

Fig. 2.

Analysis of DIG-labelled probe by agarose electrophoresis. M: molecular weight marker. LacZ (lanes 1 and 2) and neo (lanes 3 and 4) probes synthesised with (lanes 1 and 3) and without (lanes 2 and 4) DIG-labelled nucleotide. A difference in electrophoretic mobility must be observed.

Fig. 3.

Example of Southern blot results obtained with DIG-labelled probes. A, Map of a typical tm1a targeted allele [1] with position of lacZ (blue) and neo (red) probes. B, Examples of Southern blot autoradiographs obtained using either the lacZ probe (left hand side) or neo probe (right hand side) of tm1a ES cell clones. Each blot has two size standards; a small ladder (S) and a large ladder (L), with their sizes indicated in bold. The expected size (kb) of each of the visualised fragments is displayed above each of the lanes. The interpretation of each line is indicated by symbols as per the key.

Fig. 4.

Example of three projects (Cldn10, Cotl1 and Isl1) analysed by Southern blot using radioactive probes. For each project, three or four clones have been processed. A lacZ probe was used only when no acceptable 5′ restriction size (> 20 kb) was possible (Cldn10 project). For the Cotl1 project, the 3′ ApaI digest for clone 3 does not show a double band, whilst other digests do. This suggests that two bands of the same size are generated by this enzyme.