ABSTRACT
A long-standing objective of metabolic engineering has been to exogenously increase the expression of target genes. In this research, we proposed the permanent RNA replication system using DNA as a template to store genetic information in bacteria. We selected Qß phage as the RNA replication prototype and made many improvements to achieve target gene expression enhancement directly by increasing mRNA abundance. First, we identified the endogenous gene Rnc, the knockout of which significantly improved the RNA replication efficiency. Second, we elucidated the essential elements for RNA replication and optimized the system to make it more easily applicable. Combined with optimization of the host cell and the system itself, we developed a stable RNA-to-RNA replication tool to directly increase the abundance of the target mRNA and subsequently the target protein. Furthermore, it was proven efficient in enhancing the expression of specific proteins and was demonstrated to be applicable in metabolic engineering. Our system has the potential to be combined with any of the existing methods for increasing gene expression.
Subject(s)
Gene Expression Regulation , Metabolic Engineering/methods , Allolevivirus/genetics , Escherichia coli/metabolism , Escherichia coli Proteins/genetics , Luminescent Proteins/genetics , Plasmids/genetics , Plasmids/metabolism , Q beta Replicase/genetics , RNA, Messenger/metabolism , RNA, Viral/genetics , Ribonuclease III/deficiency , Ribonuclease III/geneticsABSTRACT
OBJECTIVE: RNAe is a new method that enhances protein expression at the post-transcriptional level. RNAe utility was further explored to improve endogenous protein expression. RESULTS: Transgenic mice were created by targeting RNAe to growth hormone gene into the C57/BL mouse genome by transposon mediated integration; the mice showed a heavier body weight and longer body length compared with normal mice. RNAe can also be used for gene therapy through the delivery of in vitro transcribed RNA. CONCLUSION: This study takes a further step towards applying RNAe in pharmaceutical approaches by transposon-based transgenic mice model construction and the use of in vitro transcribed RNA transfection assay.
Subject(s)
Growth Hormone/genetics , RNA, Long Noncoding/genetics , RNA, Messenger/genetics , Animals , Cell Line , Enzyme-Linked Immunosorbent Assay , Female , Flow Cytometry , Genetic Therapy/methods , HeLa Cells , Humans , Male , Mice , Mice, Transgenic , PregnancyABSTRACT
In this study, a universal protein expression enhancement RNA tool, termed RNAe, was developed by modifying a recently discovered natural long non-coding RNA. At the moment, RNAe is the only technology for gene expression enhancement, as opposed to silencing, at the post-transcriptional level. With this technology, an expression enhancement of 50-1000% is achievable, with more than 200% enhancement achieved in most cases. This work identified the sufficient and necessary element for RNAe function, which was found to be merely 300 nucleotides long and was named minRNAe. It contains a 72-nt 5' pairing sequence which determines the specificity, a 167-nt short non-pairing interspersed nuclear element (SINE) B2 sequence which enhances ribosome recruitment to the target mRNA, and a poly(A) tail, provided together on a plasmid bearing the appropriate sequences. Cellular delivery of RNAe was achieved using routine transfection. The RNAe platform was validated in several widely-used mammalian cell lines. It was proven to be efficient and flexible in specifically enhancing the expression of various endogenous and exogenous proteins of diverse functions in a dose-dependent manner. Compared to the expression-inhibitory tool RNAi, the RNAe tool has a comparable effect size, with an enhancing as opposed to inhibitory effect. One may predict that this brand new technology for enhancing the production of proteins will find wide applications in both research and biopharmaceutical production.
Subject(s)
Gene Expression Regulation , Protein Biosynthesis , Protein Engineering/methods , RNA, Long Noncoding/chemistry , Antibody Formation , Cell Line , Genetic Vectors , Green Fluorescent Proteins/analysis , Green Fluorescent Proteins/genetics , HEK293 Cells , Humans , Proteomics , RNA, Antisense/chemistry , Repetitive Sequences, Nucleic Acid , Ribosomes/metabolismABSTRACT
Cell-substrate interaction is important in tissue engineering. Vascular smooth muscle cells (VSMCs) cultured on the microgrooved surface of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx) showed a distinctive polarized morphology and a high expression level of let-7a compared with the flat substrates. LIMK2, a crucial regulator of actin dynamics, was identified as a new target of let-7a. F-Actin content on flat substrates was significantly higher than that on microgrooved ones. Either overexpression of let-7a on flat substrates or inhibited expression on microgrooved substrates can rescue the difference. In accord with actin dynamics, the expressions of contractile smooth muscle markers, such as SM22 and SMA, decreased in VSMCs cultured on microgrooved substrates compared to those on flat ones, though PHBHHx can induce the synthetic-to-contractile phenotype shift. These results indicate that microgrooved PHBHHx could enhance actin dynamics of VSMCs through let-7a-involved regulation and trigger a synthetic shift.
