Journal: International Journal of Molecular Sciences
Article Title: Allele-Specific Knockout by CRISPR/Cas to Treat Autosomal Dominant Retinitis Pigmentosa Caused by the G56R Mutation in NR2E3
doi: 10.3390/ijms22052607
Figure Lengend Snippet: Characterization of the G56R induced pluripotent stem cells (iPSC) line from the NR2E3 -adRP patient. ( A ) Bright-field microscopy of a G56R iPSC colonies. ( B ) Sanger sequencing of exon 2 of NR2E3 showing a G at cDNA position 166 in the DNA of wild type (WT) iPSC and the G>A transition at c.166 in the G56R iPSC line. ( C ) RT-PCR detection of the reprogramming vectors in RNA from non-transduced patient fibroblasts (Fibro; negative control), fibroblasts transduced with the Sendai virus vectors (Fibro + SeV; positive control) and G56R iPSCs using primers specific for either the vector backbone (SeV) or for each reprogramming cassette: polycistronic KLF4 , OCT3/4, and SOX2 (KOS), or monocistronic KLF4 and MYC . Primers for the housekeeping gene GAPDH were used as a positive control for the PCR reaction. ( D ) Karyotype analysis of the G56R iPSC line showing normal chromosomal number and structure. ( E ) qPCR analysis of the expression of the host pluripotency genes NANOG (left), OCT3/4 (middle), and LIN28A (right) in cDNA from non-transduced patient fibroblasts (Fibro; negative control), fibroblasts transduced with the Sendai virus vectors (Fibro + SeV) and G56R iPSCs. The results are expressed as mean ± SEM ( n = 3). ( F ) Immunofluorescence (IF) studies of the expression of endogenous NANOG (left), OCT3/4 (middle) and SOX2 (right) in G56R iPSCs; nuclei labelled in blue. ( G ) IF studies following embryoid body differentiation of G56R iPSCs for the expression of the germ layer markers Alpha Fetal Protein (AFP) (endoderm; left), Smooth Muscle Actin (SMA) (mesoderm; middle), and Nestin (ectoderm; right).
Article Snippet: The NR2E3 cDNA was isolated from the Clontech Human Retina QUICK-Clone cDNA pool (TaKaRa Bio Europe, Saint Germain en Laye, France) while using specific primers , and the amplicon was cloned into the pGEM-T Easy vector system (Promega), according to the manufacturer’s instructions.
Techniques: Microscopy, Sequencing, Reverse Transcription Polymerase Chain Reaction, Negative Control, Transduction, Positive Control, Plasmid Preparation, Expressing, Immunofluorescence
