High-level Transgene Expression Research Articles

Revisiting the impact of genomic hot spots: C12orf35 locus as a hot spot and engineering target.

Traditional Chinese hamster ovary (CHO) cell line development is based on random integration (RI) of transgene that causes clonal variation and subsequent large-scale clone screening. Therefore, site-specific integration (SSI) of transgenes into genomic hot spots has recently emerged as an alternative method for cell line development. However, the specific mechanisms underlying hot spot site formation remain unclear. In this study, we aimed to generate landing pad (LP) cell lines via the RI of transgenes encoding fluorescent reporter proteins flanked by recombination sites to facilitate recombinase-mediated cassette exchange. The RI-based LP cell line expressing high reporter levels with spontaneous C12orf35 locus deletion exhibited similar reporter fluorescent protein levels compared to targeted integrants with an identical reporter LP construct at the CHO genome hot spot, the C12orf35 locus. Additionally, Resf1, a C12orf35 locus gene, knockout (KO) in the RI-based LP cell line with conserved C12orf35 increased reporter expression levels, comparable to those in cell lines with C12orf35 locus disruption. These results indicate that the effect of SSI into the C12orf35 locus, a genomic hot spot, on high-level transgene expression was caused by C12orf35 disruption. In contrast to C12orf35 KO, KO at other well-known hot spot sites at specific loci of genes, including Fer1L4, Hprt1, Adgrl4, Clcc1, Dop1b, and Ddc, did not increase transgene expression. Overall, our findings suggest that C12orf35 is a promising engineering target and a hot spot for SSI-based cell line development.

Evaluation of Mono- and Bi-Functional GLOBE-Based Vectors for Therapy of β-Thalassemia by HBBAS3 Gene Addition and Mutation-Specific RNA Interference.

Therapy via the gene addition of the anti-sickling βAS3-globin transgene is potentially curative for all β-hemoglobinopathies and therefore of particular clinical and commercial interest. This study investigates GLOBE-based lentiviral vectors (LVs) for βAS3-globin addition and evaluates strategies for an increased β-like globin expression without vector dose escalation. First, we report the development of a GLOBE-derived LV, GLV2-βAS3, which, compared to its parental vector, adds anti-sickling action and a transcription-enhancing 848-bp transcription terminator element, retains high vector titers and allows for superior β-like globin expression in primary patient-derived hematopoietic stem and progenitor cells (HSPCs). Second, prompted by our previous correction of HBBIVSI-110(G>A) thalassemia based on RNApol(III)-driven shRNAs in mono- and combination therapy, we analyzed a series of novel LVs for the RNApol(II)-driven constitutive or late-erythroid expression of HBBIVSI-110(G>A)-specific miRNA30-embedded shRNAs (shRNAmiR). This included bifunctional LVs, allowing for concurrent βAS3-globin expression. LVs were initially compared for their ability to achieve high β-like globin expression in HBBIVSI-110(G>A)-transgenic cells, before the evaluation of shortlisted candidate LVs in HBBIVSI-110(G>A)-homozygous HSPCs. The latter revealed that β-globin promoter-driven designs for monotherapy with HBBIVSI-110(G>A)-specific shRNAmiRs only marginally increased β-globin levels compared to untransduced cells, whereas bifunctional LVs combining miR30-shRNA with βAS3-globin expression showed disease correction similar to that achieved by the parental GLV2-βAS3 vector. Our results establish the feasibility of high titers for LVs containing the full HBB transcription terminator, emphasize the importance of the HBB terminator for the high-level expression of HBB-like transgenes, qualify the therapeutic utility of late-erythroid HBBIVSI-110(G>A)-specific miR30-shRNA expression and highlight the exceptional potential of GLV2-βAS3 for the treatment of severe β-hemoglobinopathies.

A heat-inducible expression system for external control of gene expression in plastids.

Inducible expression systems can overcome the trade-off between high-level transgene expression and its pleiotropic effects on plant growth. In addition, they can facilitate the expression of biochemical pathways that produce toxic metabolites. Although a few inducible expression systems for the control of transgene expression in plastids have been developed, they all depend on chemical inducers and/or nuclear transgenes. Here we report a temperature-inducible expression system for plastids that is based on the bacteriophage λ leftward and rightward promoters (pL/pR) and the temperature-sensitive repressor cI857. We show that the expression of green fluorescent protein (GFP) in plastids can be efficiently repressed by cI857 under normal growth conditions, and becomes induced over time upon exposure to elevated temperatures in a light-dependent process. We further demonstrate that by introducing into plastids an expression system based on the bacteriophage T7 RNA polymerase, the temperature-dependent accumulation of GFP increased further and was ~24 times higher than expression driven by the pL/pRpromoter alone, reaching ~0.48% of the total soluble protein. In conclusion, our heat-inducible expression system provides a new tool for the external control of plastid (trans) gene expression that is cost-effective and does not depend on chemical inducers.

Integrase Defective Lentiviral Vector Promoter Impacts Transgene Expression in Target Cells and Magnitude of Vector-Induced Immune Responses.

Integrase defective lentiviral vectors (IDLVs) are a promising vaccine delivery platform given their ability to induce high magnitude and durable antigen-specific immune responses. IDLVs based on the simian immunodeficiency virus (SIV) are significantly more efficient at transducing human and simian dendritic cells (DCs) compared to HIV-based vectors, resulting in a higher expansion of antigen-specific CD8+ T cells. Additionally, IDLV persistence and continuous antigen expression in muscle cells at the injection site contributes to the durability of the vaccine-induced immune responses. Here, to further optimize transgene expression levels in both DCs and muscle cells, we generated ten novel lentiviral vectors (LVs) expressing green fluorescent protein (GFP) under different hybrid promoters. Our data show that three of the tested hybrid promoters resulted in the highest transgene expression levels in mouse DCs, monkey DCs and monkey muscle cells. We then used the three LVs with the highest in vitro transgene expression levels to immunize BALB/c mice and observed high magnitude T cell responses at 3 months post-prime. Our study demonstrates that the choice of the vector promoter influences antigen expression levels in target cells and the ensuing magnitude of T cell responses in vivo.

Combination of MAR and intron increase transgene expression of episomal vectors in CHO cells.

Previous work has shown that the EF-1α promoter of episomal vectors maintains high-level transgene expression in stably transfected Chinese-hamster ovary (CHO) cells. However, the transgene expression levels need to be further increased. Here, we first incorporated matrix attachment regions (MAR), ubiquitous chromatin opening element (UCOE), stabilizing anti repressor elements 40 (STAR 40) elements into episomal vector at different sites and orientations, and systemically assessed their effects on transgene expression in transfected CHO-K1 cells. Results showed that enhanced green fluorescent protein (eGFP) expression levels increased remarkably when MAR X-29 was inserted upstream of the promoter, followed by the insertion of MAR-1 downstream of the poly A, and the orientation had no significant effect. Moreover, MAR X-29 combined with human cytomegalovirus intron (hCMVI) yielded the highest transgene expression levels (4.52-fold). Transgene expression levels were not exclusively dependent on transgene copy numbers and were not related to the mRNA expression level. In addition, vector with MAR X-29+ hCMVI can induce herpes simplex virus thymidine kinase (HSV-TK) protein expression, and the HSV-TK protein showed a cell-killing effect and an obvious bystander effect on HCT116 cells. In conclusion, the combination of MAR X-29 and hCMV intron can achieve high efficiency transgene expression mediated by episomal vectors in CHO-K1 cells. This article is protected by copyright. All rights reserved.

A mosaic adeno-associated virus vector as a versatile tool that exhibits high levels of transgene expression and neuron specificity in primate brain

Recent emphasis has been placed on gene transduction mediated through recombinant adeno-associated virus (AAV) vector to manipulate activity of neurons and their circuitry in the primate brain. In the present study, we created a novel vector of which capsid was composed of capsid proteins derived from both of the AAV serotypes 1 and 2 (AAV1 and AAV2). Following the injection into the frontal cortex of macaque monkeys, this mosaic vector, termed AAV2.1 vector, was found to exhibit the excellence in transgene expression (for AAV1 vector) and neuron specificity (for AAV2 vector) simultaneously. To explore its applicability to chemogenetic manipulation and in vivo calcium imaging, the AAV2.1 vector expressing excitatory DREADDs or GCaMP was injected into the striatum or the visual cortex of macaque monkeys, respectively. Our results have defined that such vectors secure intense and stable expression of the target proteins and yield conspicuous modulation and imaging of neuronal activity.

Systemic gene therapy using an AAV44.9 vector rescues a neonatal lethal mouse model of propionic acidemia

Propionic acidemia (PA) is rare autosomal recessive metabolic disorder caused by defects in the mitochondrially localized enzyme propionyl-coenzyme A (CoA) carboxylase. Patientswith PA can suffer from lethal metabolic decompensation and cardiomyopathy despite current medical management, which has led to the pursuit of gene therapy as a new treatment option for patients. Here we assess the therapeutic efficacy of a recently described adeno-associated virus (AAV) capsid, AAV44.9, to deliver a therapeutic PCCA transgene in a new mouse model of propionyl-CoA carboxylase α (PCCA) deficiency generated by genome editing. Pcca-/- mice recapitulate the severe neonatal presentation of PA and manifest uniform neonatal lethality, absent PCCA expression, and increased 2-methylcitrate. A single injection of the AAV44.9 PCCA vector in the immediate newborn period, systemically delivered at a dose of 1e11 vector genome (vg)/pup but not 1e10 vg/pup, increased survival, reduced plasma methylcitrate, and resulted in high levels of transgene expression in the liver and heart in treated Pcca-/- mice. Our studies not only establish a versatile and accurate new mouse model of PA but further demonstrate that the AAV44.9 vectors may be suitable for treatment of many metabolic disorders where hepato-cardiac transduction following systemic delivery is desired, such as PA, and, by extension, fatty acid oxidation defects and glycogen storage disorders.

The effect of Drosophila attP40 background on the glomerular organization of Or47b olfactory receptor neurons.

Bacteriophage integrase-directed insertion of transgenic constructs into specific genomic loci has been widely used by Drosophila community. The attP40 landing site located on the second chromosome gained popularity because of its high inducible transgene expression levels. Here, unexpectedly, we found that homozygous attP40 chromosome disrupts normal glomerular organization of Or47b olfactory receptor neuron (ORN) class in Drosophila. This effect is not likely to be caused by the loss of function of Msp300, where the attP40 docking site is inserted. Moreover, the attP40 background seems to genetically interact with the second chromosome Or47b-GAL4 driver, which results in a similar glomerular defect. Whether the ORN phenotype is caused by the neighbouring genes around Msp300 locus in the presence of attP40-based insertions or a second unknown mutation in the attP40 background remains elusive. Our findings tell a cautionary tale about using this popular transgenic landing site, highlighting the importance of rigorous controls to rule out the attP40 landing site-associated background effects.

Transgene insertion into the plastid genome alters expression of adjacent native chloroplast genes at the transcriptional and translational levels.

In plant biotechnology and basic research, chloroplasts have been used as chassis for the expression of various transgenes. However, potential unintended side effects of transgene insertion and high-level transgene expression on the expression of native chloroplast genes are often ignored and have not been studied comprehensively. Here, we examined expression of the chloroplast genome at both the transcriptional and translational levels in five transplastomic tobacco (Nicotiana tabacum) lines carrying the identical aadA resistance marker cassette in diverse genomic positions. Although none of the lines exhibits a pronounced visible phenotype, the analysis of three lines that contain the aadA insertion in different locations within the petL-petG-psaJ-rpl33-rps18 transcription unit demonstrates that transcriptional read-through from the aadA resistance marker is unavoidable, and regularly causes overexpression of downstream sense-oriented chloroplast genes at the transcriptional and translational levels. Investigation of additional lines that harbour the aadA intergenically and outside of chloroplast transcription units revealed that expression of the resistance marker can also cause antisense effects by interference with transcription/transcript accumulation and/or translation of downstream antisense-oriented genes. In addition, we provide evidence for a previously suggested role of genomically encoded tRNAs in chloroplast transcription termination and/or transcript processing. Together, our data uncover principles of neighbouring effects of chloroplast transgenes and suggest general strategies for the choice of transgene insertion sites and expression elements to minimize unintended consequences of transgene expression on the transcription and translation of native chloroplast genes.

Alphaviruses in cancer immunotherapy.

Alphaviruses have frequently been engineered for cancer therapy, cancer immunotherapy, and cancer vaccine development. As members of self-replicating RNA viruses, alphaviruses provide high levels of transgene expression through efficient self-amplifying of their RNA genome in host cells. Alphavirus vectors can be used as recombinant viral particles or oncolytic viruses. Alternatively, either naked or nanoparticle-encapsulated RNA and DNA replicons can be utilized. In the context of cancer prevention and treatment, antitumor, cytotoxic and suicide genes have been expressed from alphavirus vectors to provide tumor regression and tumor eradication. Moreover, immunostimulatory genes such as cytokines and chemokines have been used for cancer immunotherapy approaches. Expression of tumor antigens has been applied for cancer vaccine development. Alphavirus vectors has demonstrated tumor regression and even cure in various preclinical animal models. Immunization has elicited strong immune responses and showed protection against challenges with tumor cells in animal models. Several clinical trials have confirmed good safety and tolerability of alphaviruses in cancer patients although therapeutic efficacy will still require optimization.

Therapeutic Applications for Oncolytic Self-Replicating RNA Viruses.

Self-replicating RNA viruses have become attractive delivery vehicles for therapeutic applications. They are easy to handle, can be rapidly produced in large quantities, and can be delivered as recombinant viral particles, naked or nanoparticle-encapsulated RNA, or plasmid DNA-based vectors. The self-replication of RNA in infected host cells provides the means for generating much higher transgene expression levels and the possibility to apply substantially reduced amounts of RNA to achieve similar expression levels or immune responses compared to conventional synthetic mRNA. Alphaviruses and flaviviruses, possessing a single-stranded RNA genome of positive polarity, as well as measles viruses and rhabdoviruses with a negative-stranded RNA genome, have frequently been utilized for therapeutic applications. Both naturally and engineered oncolytic self-replicating RNA viruses providing specific replication in tumor cells have been evaluated for cancer therapy. Therapeutic efficacy has been demonstrated in animal models. Furthermore, the safe application of oncolytic viruses has been confirmed in clinical trials. Multiple myeloma patients treated with an oncolytic measles virus (MV-NIS) resulted in increased T-cell responses against the measles virus and several tumor-associated antigen responses and complete remission in one patient. Furthermore, MV-CEA administration to patients with ovarian cancer resulted in a stable disease and more than doubled the median overall survival.

Culture of Human Retinal Explants for Ex Vivo Assessment of AAV Gene Delivery.

Due to the clinically established safety and efficacy profile of recombinant adeno-associated viral (rAAV) vectors, they are considered the "go to" vector for retinal gene therapy. Design of a rAAV-mediated gene therapy focuses on cell tropism, high transduction efficiency, and high transgene expression levels to achieve the lowest therapeutic treatment dosage and avoid toxicity. Human retinal explants are a clinically relevant model system for exploring these aspects of rAAV-mediated gene delivery. In this chapter, we describe an ex vivo human retinal explant culture protocol to evaluate transgene expression in order to determine the selectivity and efficacy of rAAV vectors for human retinal gene therapy.

Chloroplast genomes and GMOs. History, features and perspectives on plastid transgenesis in plant biotechnology

Chloroplasts provide life on our planet, by creating carbohydrates, amino acids, lipids and O2 through the process of photosynthesis. Two billion years ago, a eukaryotic ancestor entered symbiosis with photosynthetic cyanobacterium, giving rise to chloroplasts. During evolution, chloroplasts transferred most protein-coding genes of free-living bacteria to the nucleus, but retained a semi-autonomous genetic status their own genomes and transcription and translation machinery.
 The genetics of plastids began in 1909, when Baur and Correns described the non-Mendelian, maternal inheritance of Pelargonium leaf color: green, white and variegated. The presence DNA in chloroplasts (chDNA) was prove in the 1960s, and later have been shown, that male chDNA in zygotes is specifically degraded, assuring maternal inheritance in most plants.
 The plastid transformation was first described in the unicellular green alga Chlamydomonas reinhardtii [1]. This method of plant biotechnology is an alternative to traditional introduction of foreign DNA into the nucleus, and has a number of advantages: (a) the large copy number of chDNA offers high-level transgene expression; (b) site-specific integration of foreign genes reduces the number of transgenic lines to be evaluated; (c) the lack of gene silencing or position effects in chloroplast genomes; (d) the chloroplast employs a prokaryotic gene expression system, and allows the expression of polycistronic genes [2]. Thus, chloroplasts are becoming attractive hosts for the introduction of new agronomic traits, as well as for the biosynthesis of high-value pharmaceuticals, biomaterials and industrial enzymes.

Advances in plastid transformation for metabolic engineering in higher plants.

The plastid (chloroplast) genome of higher plants is an appealing target for metabolic engineering via genetic transformation. Although the bacterial-type plastid genome is small compared with the nuclear genome, it can accommodate large quantities of foreign genes that precisely integrate through homologous recombination. Engineering complex metabolic pathways in plants often requires simultaneous and concerted expression of multiple transgenes, the possibility of stacking several transgenes in synthetic operons makes the transplastomic approach amazing. The potential for extraordinarily high-level transgene expression, absence of epigenetic gene silencing and transgene containment due to the exclusion of plastids from pollen transmission in most angiosperm species further add to the attractiveness of plastid transformation technology. This minireview describes recent advances in expanding the toolboxes for plastid genome engineering, and highlights selected high-value metabolites produced using transplastomic plants, including artemisinin, astaxanthin and paclitaxel.

Intratumoral electroporation of a self-amplifying RNA expressing IL-12 induces antitumor effects in mouse models of cancer

Alphavirus vectors based on self-amplifying RNA (saRNA) generate high and transient levels of transgene expression and induce innate immune responses, making them an interesting tool for antitumor therapy. These vectors are usually delivered as viral particles, but it is also possible to administer them as RNA. We evaluated this possibility by in vivo electroporation of Semliki Forest virus (SFV) saRNA for local treatment of murine colorectal MC38 subcutaneous tumors. Optimization of saRNA electroporation conditions in tumors was performed using an SFV vector coding for luciferase. Then we evaluated the therapeutic potential of this approach using an SFV saRNA coding for interleukin-12 (SFV-IL-12), a proinflammatory cytokine with potent antitumor effects. Delivery of SFV-IL-12 saRNA by electroporation led to improvement in tumor control and higher survival compared with mice treated with electroporation or with SFV-IL-12 saRNA alone. The antitumor efficacy of SFV-IL-12 saRNA electroporation increased by combination with systemic PD-1 blockade. This therapy, which was also validated in a hepatocellular carcinoma tumor model, suggests that local delivery of saRNA by electroporation could be an attractive strategy for cancer immunotherapy. This approach could have easy translation to the clinical practice, especially for percutaneously accessible tumors.

Overcoming Poor Transgene Expression in the Wild-Type Chlamydomonas Chloroplast: Creation of Highly Mosquitocidal Strains of Chlamydomonas reinhardtii.

High-level expression of transgenes in the chloroplast of wild-type Chlamydomonas reinhardtii (C. reinhardtii) remains challenging for many genes (e.g., the cry toxin genes from Bacillus thuringiensis israelensis). The bottleneck is presumed to be post-transcriptional and mediated by the 5′ element and the coding region. Using 5′ elements from highly expressed photosynthesis genes such as atpA did not improve the outcome with cry11A regardless of the promoter. However, when we employed the 5′ UTR from mature rps4 mRNA with clean fusions to promoters, production of the rCry11A protein became largely promoter-dependent. The best results were obtained with the native 16S rrn promoter (−91 to −1). When it was fused to the mature 5′ rps4 UTR, rCry11A protein levels were ~50% higher than was obtained with the inducible system, or ~0.6% of total protein. This level was sufficient to visualize the 73-kDa rCry11A protein on Coomassie-stained gels of total algal protein. In addition, analysis of the expression of these transgenes by RT-PCR indicated that RNA levels roughly correlated with protein production. Live cell bioassays using the best strains as food for 3rd instar Aedes aegypti larvae showed that most larvae were killed even when the cell concentration was as low as 2 × 104 cells/mL. Finally, the results indicate that these highly toxic strains are also quite stable, and thus represent a key milestone in using C. reinhardtii for mosquito control.

Engineering and validation of a dual luciferase reporter system for quantitative and systematic assessment of regulatory sequences in Chinese hamster ovary cells

Ongoing research efforts to identify potent regulatory sequences that deliver robust and sustained transgene expression are critical for Chinese hamster ovary (CHO) cell line development technologies to meet the growing demand for recombinant proteins. Here we report the engineering and validation of a highly customizable single vector toolkit that comprises an all-in-one dual luciferase reporter system for quantitative and systematic interrogation of transcriptional regulatory sequences in transient and stable transfectants of CHO cells. To model the execution of the reporter system, we implemented a battery of known constitutive promoters including human CMV-mIE, SV40, HSV-TK, mouse PGK, human EF1α, EF1α short (EFS), human UBC, synthetic CAG, and Chinese hamster EF1α (CHEF1α). Of the nine promoters, CMV-mIE yielded the highest transcriptional activity in transient transfection settings, while CHEF1α was the strongest among a select subset of promoters in stable transfectants of CHO-DG44 pools. Remodeling the vector toolkit to build a dual fluorescent reporter system featured an alternative to bioluminescence based reporters. We infer that the findings of this study may serve as a basis to establish new vectors with weak or strong constitutive promoters. Furthermore, the modular all-in-one architecture of the reporter system proved to be a viable tool for discovering novel regulatory sequences that ensure high levels of transient and stable transgene expression in CHO and perhaps other mammalian cell lines.

A versatile transposon-based technology to generate loss- and gain-of-function phenotypes in the mouse liver

BackgroundUnderstanding the contribution of gene function in distinct organ systems to the pathogenesis of human diseases in biomedical research requires modifying gene expression through the generation of gain- and loss-of-function phenotypes in model organisms, for instance, the mouse. However, methods to modify both germline and somatic genomes have important limitations that prevent easy, strong, and stable expression of transgenes. For instance, while the liver is remarkably easy to target, nucleic acids introduced to modify the genome of hepatocytes are rapidly lost, or the transgene expression they mediate becomes inhibited due to the action of effector pathways for the elimination of exogenous DNA. Novel methods are required to overcome these challenges, and here we develop a somatic gene delivery technology enabling long-lasting high-level transgene expression in the entire hepatocyte population of mice.ResultsWe exploit the fumarylacetoacetate hydrolase (Fah) gene correction-induced regeneration in Fah-deficient livers, to demonstrate that such approach stabilizes luciferase expression more than 5000-fold above the level detected in WT animals, following plasmid DNA introduction complemented by transposon-mediated chromosomal gene transfer. Building on this advancement, we created a versatile technology platform for performing gene function analysis in vivo in the mouse liver. Our technology allows the tag-free expression of proteins of interest and silencing of any arbitrary gene in the mouse genome. This was achieved by applying the HADHA/B endogenous bidirectional promoter capable of driving well-balanced bidirectional expression and by optimizing in vivo intronic artificial microRNA-based gene silencing. We demonstrated the particular usefulness of the technology in cancer research by creating a p53-silenced and hRas G12V-overexpressing tumor model.ConclusionsWe developed a versatile technology platform for in vivo somatic genome editing in the mouse liver, which meets multiple requirements for long-lasting high-level transgene expression. We believe that this technology will contribute to the development of a more accurate new generation of tools for gene function analysis in mice.

Degron tagging of BleoR and other antibiotic-resistance genes selects for higher expression of linked transgenes and improved exosome engineering

Five antibiotic resistance (AR) genes have been used to select for transgenic eukaryotic cell lines, with the BleoR, PuroR, HygR, NeoR, and BsdR cassettes conferring resistance to zeocin, puromycin, hygromycin, geneticin/G418, and blasticidin, respectively. We recently demonstrated that each AR gene establishes a distinct threshold of transgene expression below which no cell can survive, with BleoR selecting for the highest level of transgene expression, nearly ∼10-fold higher than in cells selected using the NeoR or BsdR markers. Here, we tested the hypothesis that there may be an inverse proportionality between AR protein function and the expression of linked, transgene-encoded, recombinant proteins. Specifically, we fused each AR protein to proteasome-targeting degron tags, used these to select for antibiotic-resistant cell lines, and then measured the expression of the linked, recombinant protein, mCherry, as a proxy marker of transgene expression. In each case, degron-tagged AR proteins selected for higher mCherry expression than their cognate WT AR proteins. ER50BleoR selected for the highest level of mCherry expression, greater than twofold higher than BleoR or any other AR gene. Interestingly, use of ER50BleoR as the selectable marker translated to an even higher, 3.5-fold increase in the exosomal loading of the exosomal cargo protein, CD63/Y235A. Although a putative CD63-binding peptide, CP05, has been used to decorate exosome membranes in a technology known as “exosome painting,” we show here that CP05 binds equally well to CD63−/− cells, WT 293F cells, and CD63-overexpressing cells, indicating that CP05 may bind membranes nonspecifically. These results are of high significance for cell engineering and especially for exosome engineering.

Comprehensive analysis of plastid gene expression during fruit development and ripening of kiwifruit.

Global survey of plastid gene expression during fruit ripening in kiwifruit provides cis-elements for the futureengineering ofthe plastid genome of kiwifruit. A limitation in the application of plastid biotechnology for molecular farming is the low-level expression of transgenes in non-green plastids compared with photosynthetically active chloroplasts. Unlike other fruits, not all chloroplasts are transformed into chromoplasts during ripening of red-fleshed kiwifruit (Actinidia chinensis cv. Hongyang) fruits, which may make kiwifruit an ideal horticultural plant for recombinant protein production by plastid engineering. To identify cis-elements potentially triggering high-level transgene expression in edible tissues of the 'Hongyang' kiwifruit, here we report a comprehensive analysis of kiwifruit plastid gene transcription in green leaves and fruits at three different developmental stages. While transcripts of a few photosynthesis-related genes and most genetic system genes were substantially upregulated in green fruits compared with leaves, nearly all plastid genes were significantly downregulated at the RNA level during fruit development. Expression of a few genes remained unchanged, including psbA, the gene encoding the D1 polypeptide of photosystem II. However, PsbA protein accumulation decreased continuously during chloroplast-to-chromoplast differentiation. Analysis of post-transcriptional steps in mRNA maturation, including intron splicing and RNA editing, revealed that splicing and editing may contribute to regulation ofplastid gene expression. Altogether, 40 RNA editing sites were verified, and 5 of them were newly discovered. Taken together, this study has generated a valuable resource for the analysis of plastid gene expression and provides cis-elements for future efforts to engineer the plastid genome of kiwifruit.