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Similarly, the triple mutant Δlsp123 was generated by introduction of pEX18GM-lsp2 into Δlsp13.
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Using a U87 glioma model in SCID mice, we show that the therapeutic capacity of the new CRISPR/Cas9-engineered hAMSCs is equivalent to that of therapeutic hAMSCs generated by introduction of the same therapeutic gene by transduction with a lentiviral vector previously published by our group.
Control cell lines were generated by introduction of YFP variants (Figure 5A).
HNC45, the strain containing a deletion in hlyA locus, was generated via introduction of pCWΔhlyA [16] into a spontaneous streptomycin resistant mutant of E7946 using conjugation.
The clones ΔospC/FL/1 and ΔospC/FL/2 were generated via introduction of a full-length ospC gene carried by the shuttle vector pBBE22 into ΔospC in our previous study [18].
SL1344::kan was generated by introduction of a kanamycin resistance cassette into the downstream, non-coding region between the avrA and sitD genes of Salmonella typhimurium LT2 as previously described [22].
The clones ΔdbpAB/dbpAB/1 and ΔdbpAB/dbpAB/2, which were used as a control, were generated via introduction of a pBBE22 derivative carrying the dbpAB operon in our previous study [17], and thus expressed both dbpA and dbpB under control of their native promoter.
Transgenic plants were generated by introduction of the plant expression constructs into an Agrobacterium tumefaciens strain GV3101 and transformation was done by floral dipping (Clough and Bent, 1998).
Notably, naive human ESCs, with two active X chromosomes, have recently been generated by introduction of KLF2 and KLF4 in 2i/LIF conditions (Hanna et al., 2010), and functional cooperation between PRDM14 and KLF2 should be investigated in this context.
