Fig. 6: Loss of hSOD1 transgene copy number in G1H/Cas9.
From: Efficacy and long-term safety of CRISPR/Cas9 genome editing in the SOD1-linked mouse models of ALS
The hSOD1 transgene copy number was determined by using multiplex ligation-dependent probe amplification (MLPA). a Five mouse-specific MLPA probes were designed to hybridize to two sex chromosome-specific (Nr0b1 and SRY) and three autosomal control (Ext1, Ep300, and Crebbp) genes. Two distinct MLPA probes were designed to hybridize to intron 1 (probe 1) and intron 2 (probe 2) of hSOD1 beyond the known deleted regions. The PCR amplification products generated by each probe differ in size by at least four nucleotides (nt). Nontransgenic mouse DNA (NTg mDNA) in the b absence and c presence of human DNA (1:1) was used to illustrate hSOD1 probe specificity. hSOD1 transgene copy number in the d G1H (n = 4) and e G1H/Cas9 (n = 5) mouse lines were estimated by dividing the average peak height of hSOD1 (probe 1) by the average peak height of the three mouse control genes (e, f). This ratio was then normalized to the ratio of mouse nontransgenic DNA spiked with human DNA. Three independent MLPA reactions were performed for each condition and the standard deviation (SD) was plotted to illustrate variation. hSOD1 transgene copies in G1H were estimated to be ~30 (29.7 ± 2.31). The lowest and the highest hSOD1 transgene copies remained in G1H/Cas9 mice were estimated to be ~9 (8.6 ± 0.25) and 23 (23.3 ± 1.28), respectively.
