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. 2015 Jul 23;24(19):5524–5541. doi: 10.1093/hmg/ddv283

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Published by Oxford University Press 2015. This work is written by (a) US Government employee(s) and is in the public domain in the US.

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Figure 6. — Determination of Cre activity to deplete or replace Smn. (A) Percent recombination of Smn^F7 in motor neurons as determined by ddPCR. Motor neurons were isolated with LCM from P10-P12 mice. There was no recombination in mice that did not contain a Cre driver (0.6 ± 7.4%, n = 3). The Sox2-Cre ubiquitous driver resulted in recombination in all motor neurons (104.0 ± 1.1%, n = 3) (38). Nestin-Cre + ChAT-Cre drivers also gave complete recombination in all motor neurons (101.3 ± 1.7%, n = 3). The ChAT-Cre driver alone resulted in 88.7 ± 5.8% (n = 3, P = 0.004 versus Nestin-Cre). On average only half of all motor neurons underwent recombination with just the Nestin-Cre driver (51.3 ± 8.1%, n = 3, P = 0.004). The difference in recombination efficiencies between ChAT-Cre alone and Nestin-Cre + ChAT-Cre was not statistically significant (P = 0.274). All values are normalized to two copies of Smn intron 1. Percent recombination values were determined by multiplex ddPCR. (B) SMN protein expression as determined by western blot analysis. The alleles used are depicted on the x axis and amount of SMN relative to tubulin on the y. Note that the amount of SMN protein found upon deletion of Smn with Nestin-Cre + ChAT-Cre is not statistically different from that of the SMA sample (P = 0.782). Conversely the replacement of Smn with Nestin-Cre + ChAT-Cre is not statistically different from that of the control sample (P = 0.322) (C) The number of SMN positive gems in motor neurons. All groups are statistically different by one-way ANOVA except for SMA versus Nestin-Cre + ChAT-Cre which are not different. Thus gem counts indicate equivalent levels of SMN protein in SMA- and Smn-deletion by Nestin-Cre + ChAT-Cre. (D) Smn^+/− spinal cord section revealing many motor neurons with gems. (E) Smn^−/− (SMA) sample showing lack of gems in motor neurons. (F) Nestin-Cre Smn-deletion sample shows a reduced number of gems in motor neurons. (G) Nestin-Cre + ChAT-Cre, Smn-deletion motor neurons reveal very few gems, similar to the SMA sample (n = 3 for each group).