How can the discrepancies reported between the two NFIX-/-
strains be reconciled? Among various possible explanations, one could be an alteration of neighboring gene expression. A case in point is the sequential generation of several prion protein (PrP) knockout strains that showed profoundly different phenotypes. Only later was this variation proved to be due to the unintentional activation of another gene in the vicinity of the PrP
gene, later named Doppel
], and which was shown to be neurotoxic.
Both reports of the NFIX
] describe the deletion of the second exon, which is uniformly present in all splice variants and carries the dimerization and DNA-binding domains (Figure ). In both cases the targeting constructs were based on a λ phage library derived from the mouse strain 129/Sv, and transgenic animals carrying a single knockout allele were backcrossed to C57BL/6 mice for several generations. However, each research group used a slightly different embryonic stem (ES) cell line for making the mutation. In the case of the X-NY strain the targeting vector was electroporated into J1 ES cells, which are derived from the 129S4/SvJae strain and backcrossed to the C57BL/6 mouse strain for two to five generations. The X-Freiburg targeting construct was electroporated into CJ7 ES cells, which originate from the 129S1/Sv strain (129S1/Sv-p+Tyr+KitlSl-J
) and transgenic animals were backcrossed to C57BL/6. Driller et al
] do not specify the number of backcrossings to C57BL/6, which raises the possibility that their knockout strains, although apparently congenic with those of Campbell et al
., contain a substantial segment of ES-cell-derived chromosome still flanking the knockout allele – a 'congenic footprint'.
Figure 1 For simplicity the same structure is drawn for all four NFI genes. (a) The organization of the NFI genes. They can all use an alternative exon 1, here denoted as a single box labeled 1a/1b. The DNA-binding and dimerization domains are located in exon (more ...)
In a study of congenic knockouts at another gene, Schalkwyk et al
] found that at least 10 genes across 40 Mb around the targeted locus show differences in expression in the different knockout strains, due to the congenic footprint effect. Genome-wide analysis of gene expression in different tissues of knockout animals by microarray profiling also indicates that a significant proportion of changes are found in the proximity of the targeted gene [10
]. This little excursion into the theory of induced mutation experiments does not seem so trivial in the light of several studies describing corpus callosum defects in the 129/Sv strain itself [12
], which vary between 129 substrains studied [14
]. Callosal agenesis is one of the phenotypic features ascribed to the X-Freiburg strain, while at the same time complete callosal agenesis was not seen in X-NY strain. The locus (or loci) responsible for callosal agenesis in the 129/Sv strain is not characterized and it is not unreasonable to speculate that such a region might be present in the proximity of NFIX
in the X-Freiburg strain, whereas in the X-NY strain this locus had been removed by outbreeding.
Another possible source of variation emanates from the targeting strategy used. Campbell et al
. completely deleted the second exon along with proximal parts of neighboring introns, whereas Driller et al
. replaced the second exon with a coding sequence of the LacZ
gene fused to a coding sequence of NFIX
(Figure ). In this regard, a comparison of all the available NFI
gene knockouts is perhaps more informative (Figure ). An intriguing feature that emerges from this comparison is that mice in which the 3' splice acceptor of the first intron is removed somehow have a milder phenotype. Without further experimental evidence it is difficult to explain this observation, which could be purely coincidental. Formation of an alternatively spliced gene variant (which was not looked for), as with the activation of Doppel
], is one possibility. Alternatively, the fusion of the first few amino acids of NFIX (or NFIB) to LacZ might lead to a toxic gain-of-function protein. The peptide in question is quite short but even so could endow the fusion protein with toxic properties. This hypothesis is rather easy to test by overexpressing recombinant NFIXexon1
-LacZ protein in glial or neuronal cells.
A full explanation of these intriguing phenotypes will require experimental testing and a proper analysis of the ideas put forward here, as well as other possibilities. Thorough analysis of all available knockouts might reveal surprising new functions of NFI proteins and further enhance our understanding of their biological functions.