Improving gene targeting efficiency on pig IGF2 mediated by ZFNs and CRISPR/Cas9 by using SSA reporter system.

IGF2 (Insulin-like growth factor 2) is a major growth factor affecting porcine fetal and postnatal development. We propose that the precise modification of IGF2 gene of Chinese indigenous pig breed--Lantang pig by genome editing technology could reduce its backfat thickness, and increase its lean meat content. Here, we tested the genome editing activities of zinc finger nucleases (ZFNs) and CRISPR/Cas9 system on IGF2 gene in the Lantang porcine fetal fibroblasts (PEF). The results indicated that CRISPR/Cas9 presented cutting efficiency up to 9.2%, which was significantly higher than that generated by ZFNs with DNA cutting efficiency lower than 1%. However, even by using CRISPR/Cas9, the relatively lower percentage of genetically modified cells in the transfected population was not satisfied for somatic nuclear transfer (SCNT). Therefore, we used a SSA (Single-strand annealing) reporter system to enrich genetically modified cells induced by ZFN or CRISPR/Cas9. T7 endonuclease I assay revealed that this strategy improved genome editing activity of CRISPR/Cas9 by 5 folds, and was even more effective for improving genome editing efficiency of ZFN.

[Infrared spectroscopy investigation on conformational change of silk fiber induced by gaseous organic acids existing in museum].

Study on the impact of pollutants on cultural materials in storing or displaying micro-environment in museum is considered as very important for the preservation of cultural relics and its aging prevention. This paper applied the Fourier transform infrared (attenuated total reflection) technique to assess silk structural changes under volatile organic acids (formic acid/acetic acid), which usually come from decorative materials emission and commonly exist in the surface or around cultural materials. The focus of this work was on investigating the changes of peptide bond in the area of amide I-amide III, as well as the peptide chains (GlyAla), characteristic region. The structural and conformational changes in silk fiber treated with gaseous formic and acetic acid were assessed. The results indicate that both the gaseous acids can weaken the intermolecular hydrogen bond in fiber peptide, based on the spectral changes in the increased intensity of amide I (1 617 cm(-1)), the narrowing amide II peak (1 515 cm(-1)), the increased intensity of random coil conformation in amide III peak (1 230 cm(-1)), and the decreased fiber crystallinity as well. The obvious secondary structural conformation occurred when the concentration of gaseous formic acid reached 8.1 mg x m(-3) in simulated environment. The conformational transformation was supported by the observation of the rapidly reduced random coil conformation, the increased short peptide chains (GlyAla)n with beta-sheet conformation characteristic peak (1 000, 975 cm(-1)), and the enhanced fiber crystallinity degree as well. In contrast, gaseous acetic acid has less impact on the amide I and amide II bond based on the spectral changes, but it did promote random coil conformation and decreased fiber crystallinity. This work also provides a potential application of the infrared spectroscopy in non-destructive investigation of silk in-situ.