Light it up: highly efficient multigene delivery in mammalian cells

Multigene delivery and expression systems are emerging as key technologies for many applications in contemporary biology. We have developed new methods for multigene delivery and expression in eukaryotic hosts for a variety of applications, including production of protein complexes for structural biology and drug development, provision of multicomponent protein biologics, and cell-based assays. We implemented tandem recombineering to facilitate rapid generation of multicomponent gene expression constructs for efficient transformation of mammalian cells, resulting in homogenous cell populations. Analysis of multiple parameters in living cells may require co-expression of fluorescently tagged sensors simultaneously in a single cell, at defined and ideally controlled ratios. Our method enables such applications by overcoming currently limiting challenges. Here, we review recent multigene delivery and expression strategies and their exploitation in mammalian cells. We discuss applications in drug discovery assays, interaction studies, and biologics production, which may benefit in the future from our novel approach.     

Regulated multicistronic expression technology for mammalian metabolic engineering

Contemporary basic research is rapidly revealing increasingly complex molecular regulatory networks which are often interconnected via key signal integrators. These connections among regulatory and catalytic networks often frustrate bioengineers as promising metabolic engineering strategies are bypassed by compensatory metabolic responses or cause unexpected, undesired outcomes such as apoptosis, product protein degradation or inappropriate post- translational modification. Therefore, for metabolic engineering to achieve greater success in mammalian cell culture processes and to become important for future applications such as gene therapy and tissue engineering, this technology must be enhanced to allow simultaneous, in cases conditional, reshaping of metabolic pathways to access difficult-to-attain cell states. Recent advances in this new territory of multigene metabolic engineering are intimately linked to the development of multicistronic expression technology which allows the simultaneous, and in some cases, regulated expression of several genes in mammalian cells. Here we review recent achievements in multicistronic expression technology in view of multigene metabolic engineering. This revised version was published online in August 2006 with corrections to the Cover Date.     

Viral Cre-LoxP tools aid genome engineering in mammalian cells

Background

Targeted nucleases have transformed genome editing technology, providing more efficient methods to make targeted changes in mammalian genome. In parallel, there is an increasing demand of Cre-LoxP technology for complex genome manipulation such as large deletion, addition, gene fusion and conditional removal of gene sequences at the target site. However, an efficient and easy-to-use Cre-recombinase delivery system remains lacking.

Results

We designed and constructed two sets of expression vectors for Cre-recombinase using two highly efficient viral systems, the integrative lentivirus and non-integrative adeno associated virus. We demonstrate the effectiveness of those methods in Cre-delivery into stably-engineered HEK293 cells harboring LoxP-floxed red fluorescent protein (RFP) and puromycin (Puro) resistant reporters. The delivered Cre recombinase effectively excised the floxed RFP-Puro either directly or conditionally, therefore validating the function of these molecular tools. Given the convenient options of two selections markers, these viral-based systems offer a robust and easy-to-use tool for advanced genome editing, expanding complicated genome engineering to a variety of cell types and conditions.

Conclusions

We have developed and functionally validated two viral-based Cre-recombinase delivery systems for efficient genome manipulation in various mammalian cells. The ease of gene delivery with the built-in reporters and inducible element enables live cell monitoring, drug selection and temporal knockout, broadening applications of genome editing.

     

CRISPR-based self-cleaving mechanism for controllable gene delivery in human cells

Controllable gene delivery via vector-based systems remains a formidable challenge in mammalian synthetic biology and a desirable asset in gene therapy applications. Here, we introduce a methodology to control the copies and residence time of a gene product delivered in host human cells but also selectively disrupt fragments of the delivery vehicle. A crucial element of the proposed system is the CRISPR protein Cas9. Upon delivery, Cas9 guided by a custom RNA sequence cleaves the delivery vector at strategically placed targets thereby inactivating a co-expressed gene of interest. Importantly, using experiments in human embryonic kidney cells, we show that specific parameters of the system can be adjusted to fine-tune the delivery properties. We envision future applications in complex synthetic biology architectures, gene therapy and trace-free delivery.     

Advances in CRISPR-Cas9 genome engineering: lessons learned from RNA interference

The discovery that the machinery of the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas9 bacterial immune system can be re-purposed to easily create deletions, insertions and replacements in the mammalian genome has revolutionized the field of genome engineering and re-invigorated the field of gene therapy. Many parallels have been drawn between the newly discovered CRISPR-Cas9 system and the RNA interference (RNAi) pathway in terms of their utility for understanding and interrogating gene function in mammalian cells. Given this similarity, the CRISPR-Cas9 field stands to benefit immensely from lessons learned during the development of RNAi technology. We examine how the history of RNAi can inform today''s challenges in CRISPR-Cas9 genome engineering such as efficiency, specificity, high-throughput screening and delivery for in vivo and therapeutic applications.     

Overexpression of membrane proteins in mammalian cells for structural studies

Abstract

The number of structures of integral membrane proteins from higher eukaryotes is steadily increasing due to a number of innovative protein engineering and crystallization strategies devised over the last few years. However, it is sobering to reflect that these structures represent only a tiny proportion of the total number of membrane proteins encoded by a mammalian genome. In addition, the structures determined to date are of the most tractable membrane proteins, i.e., those that are expressed functionally and to high levels in yeast or in insect cells using the baculovirus expression system. However, some membrane proteins that are expressed inefficiently in these systems can be produced at sufficiently high levels in mammalian cells to allow structure determination. Mammalian expression systems are an under-used resource in structural biology and represent an effective way to produce fully functional membrane proteins for structural studies. This review will discuss examples of vertebrate membrane protein overexpression in mammalian cells using a variety of viral, constitutive or inducible expression systems.     

Dissecting and engineering metabolic and regulatory networks of thermophilic bacteria for biofuel production

Interest in thermophilic bacteria as live-cell catalysts in biofuel and biochemical industry has surged in recent years, due to their tolerance of high temperature and wide spectrum of carbon-sources that include cellulose. However their direct employment as microbial cellular factories in the highly demanding industrial conditions has been hindered by uncompetitive biofuel productivity, relatively low tolerance to solvent and osmic stresses, and limitation in genome engineering tools. In this work we review recent advances in dissecting and engineering the metabolic and regulatory networks of thermophilic bacteria for improving the traits of key interest in biofuel industry: cellulose degradation, pentose–hexose co-utilization, and tolerance of thermal, osmotic, and solvent stresses. Moreover, new technologies enabling more efficient genetic engineering of thermophiles were discussed, such as improved electroporation, ultrasound-mediated DNA delivery, as well as thermo-stable plasmids and functional selection systems. Expanded applications of such technological advancements in thermophilic microbes promise to substantiate a synthetic biology perspective, where functional parts, module, chassis, cells and consortia were modularly designed and rationally assembled for the many missions at industry and nature that demand the extraordinary talents of these extremophiles.     

2013合成生物学专刊序言

合成生物学目前在全球得到迅猛发展。在此专刊中,综述了一些相关技术在合成生物学领域的进展,其中有:链霉菌无痕敲除方法、基因合成技术、DNA组装新方法、最小化基因组的方法及分析、合成生物系统的组合优化。也讨论了应用合成生物学策略优化光合蓝细菌底盘、产溶剂梭菌分子遗传操作技术、蛋白质预算(Protein budget)作为合成生物学的成本标尺。最后,用几个例子说明了合成生物学的应用,包括复杂天然产物合成人工生物系统的设计与构建、微生物木糖代谢途径改造制备生物基化学品以及构建酿酒酵母工程菌合成香紫苏醇。     

New Applications for Phage Integrases

Within the last 25 years, bacteriophage integrases have rapidly risen to prominence as genetic tools for a wide range of applications from basic cloning to genome engineering. Serine integrases such as that from ?C31 and its relatives have found an especially wide range of applications within diverse micro-organisms right through to multi-cellular eukaryotes. Here, we review the mechanisms of the two major families of integrases, the tyrosine and serine integrases, and the advantages and disadvantages of each type as they are applied in genome engineering and synthetic biology. In particular, we focus on the new areas of metabolic pathway construction and optimization, biocomputing, heterologous expression and multiplexed assembly techniques. Integrases are versatile and efficient tools that can be used in conjunction with the various extant molecular biology tools to streamline the synthetic biology production line.     

TEV protease-facilitated stoichiometric delivery of multiple genes using a single expression vector

Delivery and expression of multiple genes is an important requirement in a range of applications such as the engineering of synthetic signaling pathways and the induction of pluripotent stem cells. However, conventional approaches are often inefficient, nonstoichiometric and may limit the maximum number of genes that can be simultaneously expressed. We here describe a versatile approach for multiple gene delivery using a single expression vector by mimicking the protein expression strategy of RNA viruses. This was accomplished by first expressing the genes together with TEV protease as a single fusion protein, then proteolytically self-cleaving the fusion protein into functional components. To demonstrate this method in E. coli cells, we analyzed the translation products using SDS-PAGE and showed that the fusion protein was efficiently cleaved into its components, which can then be purified individually or as a binding complex. To demonstrate this method in mammalian cells, we designed a differential localization scheme and used live cell imaging to observe the distinctive subcellular targeting of the processed products. We also showed that the stoichiometry of the processed products was consistent and corresponded with the frequency of appearance of their genes on the expression vector. In summary, the efficient expression and separation of up to three genes was achieved in both E. coli and mammalian cells using a single TEV protease self-processing vector.     

Lentivirus-delivered stable gene silencing by RNAi in primary cells

Genome-wide genetic approaches have proven useful for examining pathways of biological significance in model organisms such as Saccharomyces cerevisiae, Drosophila melanogastor, and Caenorhabditis elegans, but similar techniques have proven difficult to apply to mammalian systems. Although manipulation of the murine genome has led to identification of genes and their function, this approach is laborious, expensive, and often leads to lethal phenotypes. RNA interference (RNAi) is an evolutionarily conserved process of gene silencing that has become a powerful tool for investigating gene function by reverse genetics. Here we describe the delivery of cassettes expressing hairpin RNA targeting green fluorescent protein (GFP) using Moloney leukemia virus-based and lentivirus-based retroviral vectors. Both transformed cell lines and primary dendritic cells, normally refractory to transfection-based gene transfer, demonstrated stable silencing of targeted genes, including the tumor suppressor gene TP53 in normal human fibroblasts. This report demonstrates that both Moloney leukemia virus and lentivirus vector-mediated expression of RNAi can achieve effective, stable gene silencing in diverse biological systems and will assist in elucidating gene functions in numerous cell types including primary cells.     

Production in Saccharomycescerevisiae of MS2 virus-like particles packaging functional heterologous mRNAs

Recently, DNA bacteriophages (M13, lambda) have been genetically engineered to transfer genes into mammalian cells. Although efficiencies observed are still relatively low, this opens the possibility of using these viruses as a new class of transfection agents not only for fundamental research purposes but also in gene therapy protocols or in other applications like vaccination. In this respect, it has been shown that a lambda bacteriophage engineered to express the hepatitis B surface antigen in mammalian cells could elicit an immune response against this antigen in mice and rabbits without any specific targeting of the bacteriophage. These impressive results would be even more encouraging if they could be obtained with an RNA bacteriophage, as RNA vaccines are preferred over DNA vaccines for safety reasons. Up to now, RNA bacteriophages have never been engineered for gene delivery. In this paper, we have sought to determine whether such a vector could be obtained by engineering the RNA bacteriophage MS2. We show that MS2 can be produced as virus-like particles (VLPs) in Saccharomyces cerevisiae and is able to package functional heterologous mRNAs, provided that these mRNAs contain the MS2 packaging sequence. For instance, linking the MS2 packaging sequence to the human growth hormone (hGH) mRNA enabled the packaging of this particular mRNA in MS2 VLPs. Functionality in eukaryotic systems of packaged mRNAs was confirmed by showing that mRNAs purified from VLPs can be efficiently translated in vitro and in cell cultures. The high stability of MS2 could, therefore, make MS2 VLPs a very powerful carrier for RNA vaccines.     

Efficient expression by an alphavirus replicon of a functional ribozyme targeted to human immunodeficiency virus type 1.

Intracellular applications of ribozymes have been limited partly by the availability of suitable high-expression systems. For RNA effectors, consideration of an RNA virus vector system for delivery and expression is reasonable. We show that alphavirus replicons can be highly efficient nonintegrating ribozyme-expressing vectors. Using a hammerhead ribozyme targeted to a highly conserved sequence in the U5 region of the human immunodeficiency virus type 1 (HIV-1) long terminal repeat, we demonstrate that a full-length 8.3-kb Semliki Forest virus ribozyme (SFVRz) chimeric RNA maintains catalytic activity. SFVRz is packaged into viral particles, and these particles transduce mammalian cells efficiently. SFVRz-transduced BHK cells were found to produce large amounts of genomic and subgenomic forms of ribozyme-containing RNAs that are functional in cleaving a U5-tagged mRNA. The RNase protection assay shows that HIV-1 U5-chloramphenicol acetyltransferase mRNA expressed intracellularly from an RNA polymerase II promoter is quantitatively eliminated in SFVRz-transduced BHK cells.     

Protein and RNA engineering to customize microbial molecular reporting

Nature takes advantage of the malleability of protein and RNA sequence and structure to employ these macromolecules as molecular reporters whose conformation and functional roles depend on the presence of a specific ligand (an "effector" molecule). By following nature's example, ligand-responsive proteins and RNA molecules are now routinely engineered and incorporated into customized molecular reporting systems (biosensors). Microbial small-molecule biosensors and endogenous molecular reporters based on these sensing components find a variety of applications that include high-throughput screening of biosynthesis libraries, environmental monitoring, and novel gene regulation in synthetic biology. Here, we review recent advances in engineering small-molecule recognition by proteins and RNA and in coupling in vivo ligand binding to reporter-gene expression or to allosteric activation of a protein conferring a detectable phenotype. Emphasis is placed on microbial screening systems that serve as molecular reporters and facilitate engineering the ligand-binding component to recognize new molecules.     

An EBV‐based plasmid can replicate and maintain in stem cells

Viral vectors have a wide range of applications in biology, particularly in gene therapy. Based on their integration capacity, viral vectors are classified as either integrating or non‐integrating vectors. Although integrating vectors, such as lentivectors, have the ability to direct prolonged expression of exogenous genes, manipulation of the host genome is an inappropriate feature of these gene delivery tools. Non‐integrating vectors, such as episomal replicating plasmids, can replicate and persist in host cells for long periods without any chromosomal interruption. These advantages made them good tools for gene induction purposes in gene therapy and basic studies. Due to the necessity of gene induction in stem cells for study of mammalian development and targeted differentiation, the use of integrating vectors for prolonged expression of genes of interest has been developed. Application of replicating plasmids can overcome some drawbacks associated with integrating vectors, although replication and maintenance of these plasmids can differ between cell types. Previously, it has been shown that such plasmids can be maintained in human embryonic stem cells for more than one month, but the rate of the plasmid replication during the host cell cycle has not been elucidated. In the present study, we showed that an EBV‐based plasmid can replicate simultaneously with host in pluripotent and multipotent human and mouse stem cells and can be sustained for long time periods in dividing cells. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:1579–1585, 2015     

Delivery of shRNA using gold nanoparticle-DNA oligonucleotide conjugates as a universal carrier

The efficient delivery of nucleic acids into mammalian cells is a central aspect of research involving cell biology and medical applications, including the clinical treatment of genetic disorders. We report an efficient small hairpin RNA (shRNA) delivery system that utilizes a single species of gold nanoparticle-DNA oligonucleotide conjugate (AuNP-DNA oligo) as a universal carrier. In vitro synthesized shRNA that is specific to the p53 gene was efficiently delivered into HEK293 and HeLa human cell lines using an AuNP-DNA oligo. The delivery resulted in an 80-90% knockdown of p53 expression. The same AuNP-DNA oligo was also efficient for the delivery of another shRNA, which is specific to the Mcl-1 gene, as well as the repression of MCL-1 expression. The knockdown efficiency of shRNA that was delivered using an AuNP-DNA oligo was comparable with that of a liposome-based shRNA delivery method. Our results offer an alternate delivery system for shRNA that can be used on any gene of interest.     

Lentiviral vectors encoding tetracycline-dependent repressors and transactivators for reversible knockdown of gene expression: a comparative study

Background  

RNA interference (RNAi)-mediated by the expression of short hairpin RNAs (shRNAs) has emerged as a powerful experimental tool for reverse genetic studies in mammalian cells. A number of recent reports have described approaches allowing regulated production of shRNAs based on modified RNA polymerase II (Pol II) or RNA polymerase III (Pol III) promoters, controlled by drug-responsive transactivators or repressors such as tetracycline (Tet)-dependent transactivators and repressors. However, the usefulness of these approaches is often times limited, caused by inefficient delivery and/or expression of shRNA-encoding sequences in target cells and/or poor design of shRNAs sequences. With a view toward optimizing Tet-regulated shRNA expression in mammalian cells, we compared the capacity of a variety of hybrid Pol III promoters to express short shRNAs in target cells following lentivirus-mediated delivery of shRNA-encoding cassettes.