Cell and Tissue Journal

Abstract

Abstract Aim: In this study, the alteration of the Leucine-rich repeat kinase 2 (LRRK2) gene, as the most important gene involved in Parkinson's disease, was investigated using CRISPR-Cas editing technology.
Material and methods: Cloning the guide molecules of interest was performed using of gRNA CRISPR cloning kit (Catalog No. C8324K, Zaver Zist Azema). Two sets of forward and reversed gRNA oligonucleotides for exon 41 of the LRRK2 gene were designed using the CHOPCHOP CRISPR guide gRNA designing web software. Double-stranded DNA molecules were made according to standard instructions in the laboratory. 20 base-pair DNA segments were used to generate RNA-Cas-9 enzyme complex and then ligated into the Px459 CRISPR eukaryotic expression vector using Fermentas DNA ligase enzyme. Recombinant plasmids were transformed into E. coli DH5a host competent cells using of heat shock method.
Results: findings by multiple PCR experiments and also DNA sequencing blast analysis showed successful cloning the segments of interest into CRISPR plasmids. Clear PCR bands were in agreement with expected values and DNA sequencing results showed 100% similarity. 
Conclusion: According to the results, development of CRISPR vector system may be used as a wide and powerful tool for editing and alteration of many encountered genes in different diseases such as Parkinson's disease.

Abstract Aim: The purpose of this study was to identify LEAFY homologous genes in a type of nightshade (Solanum villosum Mill.).
Material and Methods: Total RNA was isolated from shoot apical meristem tissue of S. villosum and was used for First-strand cDNA synthesis. The specific primers were designed based on nucleotide sequence alignment of LEAFY homologous genes from other plants and were used in RT-PCR. The RT-PCR products were purified and sequenced.
Results: The results indicated amplification of 540 nucleotides fragment, and named SvLFY. Comparison of SvLFY deduced protein sequences with LEAFY homologous proteins from other species showed high similarity between this fragment and in other C-terminal regions.
Conclusion: These results suggest that SvLFY may play a similar role as LEAFY in flower development and could act as a flower meristem identity gene in Solanum villosum Mill.