Acceptor Vector Research Articles

MultiGreen: A multiplexing architecture for GreenGate cloning.

Genetic modification of plants fundamentally relies upon customized vector designs. The ever-increasing complexity of transgenic constructs has led to increased adoption of modular cloning systems for their ease of use, cost effectiveness, and rapid prototyping. GreenGate is a modular cloning system catered specifically to designing bespoke, single transcriptional unit vectors for plant transformation-which is also its greatest flaw. MultiGreen seeks to address GreenGate's limitations while maintaining the syntax of the original GreenGate kit. The primary limitations MultiGreen addresses are 1) multiplexing in series, 2) multiplexing in parallel, and 3) repeated cycling of transcriptional unit assembly through binary intermediates. MultiGreen efficiently concatenates bespoke transcriptional units using an additional suite of level 1acceptor vectors which serve as an assembly point for individual transcriptional units prior to final, level 2, condensation of multiple transcriptional units. Assembly with MultiGreen level 1 vectors scales at a maximal rate of 2*⌈log6n⌉+3 days per assembly, where n represents the number of transcriptional units. Further, MultiGreen level 1 acceptor vectors are binary vectors and can be used directly for plant transformation to further maximize prototyping speed. MultiGreen is a 1:1 expansion of the original GreenGate architecture's grammar and has been demonstrated to efficiently assemble plasmids with multiple transcriptional units. MultiGreen has been validated by using a truncated violacein operon from Chromobacterium violaceum in bacteria and by deconstructing the RUBY reporter for in planta functional validation. MultiGreen currently supports many of our in-house multi transcriptional unit assemblies and will be a valuable strategy for more complex cloning projects.

Rapid Assembly and Prototyping of Biocatalytic Virus-like Particle Nanoreactors.

Protein cages are attractive as molecular scaffolds for the fundamental study of enzymes and metabolons and for the creation of biocatalytic nanoreactors for in vitro and in vivo use. Virus-like particles (VLPs) such as those derived from the P22 bacteriophage capsid protein make versatile self-assembling protein cages and can be used to encapsulate a broad range of protein cargos. In vivo encapsulation of enzymes within VLPs requires fusion to the coat protein or a scaffold protein. However, the expression level, stability, and activity of cargo proteins can vary upon fusion. Moreover, it has been shown that molecular crowding of enzymes inside VLPs can affect their catalytic properties. Consequently, testing of numerous parameters is required for production of the most efficient nanoreactor for a given cargo enzyme. Here, we present a set of acceptor vectors that provide a quick and efficient way to build, test, and optimize cargo loading inside P22 VLPs. We prototyped the system using a yellow fluorescent protein and then applied it to mevalonate kinases (MKs), a key enzyme class in the industrially important terpene (isoprenoid) synthesis pathway. Different MKs required considerably different approaches to deliver maximal encapsulation as well as optimal kinetic parameters, demonstrating the value of being able to rapidly access a variety of encapsulation strategies. The vector system described here provides an approach to optimize cargo enzyme behavior in bespoke P22 nanoreactors. This will facilitate industrial applications as well as basic research on nanoreactor-cargo behavior.

Frequency and diversity of Stenotrophomonas spp. carrying blaKPC in recreational coastal waters

Stenotrophomonas can survive in a wide range of environments and is considered an opportunistic pathogen. Because of its intrinsic resistance to beta-lactams, this genus is considered irrelevant in studies addressing the environmental spread of antimicrobial resistance genes of medical importance. Consequently, studies on environmental Stenotrophomonas carrying acquired carbapenemase-encoding genes are scarce, though not inexistent. Here, we investigated the frequency and diversity of Stenotrophomonas spp. carrying genes encoding carbapenemases of medical relevance in coastal waters with distinct pollution degrees over one year. Among 319 isolates recovered, 220 (68.9%) showed blaKPC. The frequency of blaKPC-positive Stenotrophomonas spp. was not correlated with thermotolerant counts in coastal waters evaluated. All isolates were susceptible to minocycline, levofloxacin, and trimethoprim-sulfamethoxazole. PFGE typing of 101 blaKPC-positive isolates revealed 55 pulsotypes with 5 subtypes, all of which carried the blaKPC-2 variant. Interspecies differentiation of pulsotypes’ representatives revealed 55 isolates belonging to the S. maltophilia complex (91.7%) and 5 S. acidaminiphila (8.3%). The blaKPC-2 gene was more frequently harbored on transposable elements found in enterobacteria of clinical origin, especially Tn4401b. Even though beta-lactams are no therapeutic options to treat Stenotrophomonas infections, the occurrence of a highly relevant antimicrobial resistance determinant harbored on mobile genetic elements in a diverse collection of these ubiquitous microorganisms is noteworthy. Therefore, Stenotrophomonas may act as acceptor, stable reservoirs, and potential vectors of antimicrobial resistance in environmental settings, especially aquatic matrices, and should not be neglected.

CyanoGate: A Modular Cloning Suite for Engineering Cyanobacteria Based on the Plant MoClo Syntax.

Recent advances in synthetic biology research have been underpinned by an exponential increase in available genomic information and a proliferation of advanced DNA assembly tools. The adoption of plasmid vector assembly standards and parts libraries has greatly enhanced the reproducibility of research and the exchange of parts between different labs and biological systems. However, a standardized modular cloning (MoClo) system is not yet available for cyanobacteria, which lag behind other prokaryotes in synthetic biology despite their huge potential regarding biotechnological applications. By building on the assembly library and syntax of the Plant Golden Gate MoClo kit, we have developed a versatile system called CyanoGate that unites cyanobacteria with plant and algal systems. Here, we describe the generation of a suite of parts and acceptor vectors for making (1) marked/unmarked knock-outs or integrations using an integrative acceptor vector, and (2) transient multigene expression and repression systems using known and previously undescribed replicative vectors. We tested and compared the CyanoGate system in the established model cyanobacterium Synechocystis sp. PCC 6803 and the more recently described fast-growing strain Synechococcus elongatus UTEX 2973. The UTEX 2973 fast-growth phenotype was only evident under specific growth conditions; however, UTEX 2973 accumulated high levels of proteins with strong native or synthetic promoters. The system is publicly available and can be readily expanded to accommodate other standardized MoClo parts to accelerate the development of reliable synthetic biology tools for the cyanobacterial community.

Modulating the Expression Strength of the Baculovirus/Insect Cell Expression System: A Toolbox Applied to the Human Tumor Suppressor SMARCB1/SNF5.

The human tumor suppressor SMARCB1/INI1/SNF5/BAF47 (SNF5) is a core subunit of the multi-subunit ATP-dependent chromatin remodeling complex SWI/SNF, also known as Brahma/Brahma-related gene 1 (BRM/BRG1)-associated factor (BAF). Experimental studies of SWI/SNF are currently considerably limited by the low cellular abundance of this complex; thus, recombinant protein production represents a key to obtain the SWI/SNF proteins for molecular and structural studies. While the expression of mammalian proteins in bacteria is often difficult, the baculovirus/insect cell expression system can overcome limitations of prokaryotic expression systems and facilitate the co-expression of multiple proteins. Here, we demonstrate that human full-length SNF5 tagged with a C-terminal 3 × FLAG can be expressed and purified from insect cell extracts in monomeric and dimeric forms. To this end, we constructed a set of donor and acceptor vectors for the expression of individualproteins and protein complexes in the baculovirus/insect cell expression system under the control of a polyhedrin (polh), p10, or a minimal Drosophila melanogaster Hsp70 promoter. We show that the SNF5 expression level could be modulated by the selection of the promoter used to control expression. The vector set also comprises vectors that encode a 3 × FLAG tag, Twin-Strep tag, or CBP-3 × FLAG-TEV-ProteinA triple tag to facilitate affinity selection and detection. By gel filtration and split-ubiquitin assays, we show that human full-length SNF5 has the ability to self-interact. Overall, the toolbox developed herein offers the possibility to flexibly select the promoter strength as well as the affinity tag and is suggested to advance the recombinant expression of chromatin remodeling factors and other challenging proteins.

Inhibition of v-rel-Induced Oncogenesis through microRNA Targeting.

Several studies have shown that microRNA-targeting is an effective strategy for the selective control of tissue-tropism and pathogenesis of both DNA and RNA viruses. However, the exploitation of microRNA-targeting for the inhibition of transformation by oncogenic viruses has not been studied. The v-rel oncoprotein encoded by reticuloendotheliosis virus T strain (Rev-T) is a member of the rel/NF-κB family of transcription factors capable of transforming primary chicken spleen and bone marrow cells. Here, by engineering the target sequence of endogenous microRNA miR-142 downstream of the v-rel gene in a Replication-Competent ALV (avian leukosis virus) long terminal repeat (LTR) with a splice acceptor (RCAS) vector and using a v-rel-induced transformation model of chicken embryonic splenocyte cultures, we show that hematopoietic-specific miR-142 can inhibit the v-rel-induced transformation, and that this inhibition effect is due to the silencing of v-rel expression. The data supports the idea that microRNA-targeting can be used to inhibit viral oncogene-induced oncogenesis.

In Vivo Genome Editing in Neonatal Mouse Liver Preferentially Utilizes Homology Directed Repair

Genome editing has the potential to provide long-term therapeutic gene expression in vivo. We have previously demonstrated efficient editing in a mouse model of hemophilia B through liver-directed adeno-associated viral vector (AAV) delivery of a zinc finger nuclease (ZFN) pair and a corrective donor. We determined that homology is not necessary to achieve efficient levels of genome editing in adult mice, consistent with the fact that quiescent cells, including adult hepatocytes, are not thought to be amenable to homology directed repair (HDR). As a consequence of the donor containing a splice acceptor, both HDR and homology independent vector integration are capable of driving human factor 9 (hF.IX) expression. In this study we sought to determine whether hF.IX expression in mice treated as neonates, undergoing substantial hepatocyte proliferation, is predominantly the result of HDR or homology independent genome editing. Provided the efficacy is not substantially reduced, an HDR dependent approach would impose additional constraints on targeting.Treatment of neonatal hF9mut mice (harboring the ZFN target site) with 1x1011 vg AAV8-ZFN and 5x1011 vg AAV8-Donor via retro-orbital injection resulted in a drastic difference in hF.IX expression between donors with and without homology 10 weeks post injection (Homology: 1531 ± 174.5 ng/mL vs. No-homology: 146.1 ± 5.8 ng/mL; n=12 and 7, respectively). We next asked whether HDR could be stimulated even more specifically through the induction of DNA single strand breaks at the target site. We treated neonatal mice with homologous or non-homologous donors, as well as ZFNs or ZFNickases (in which one FokI nuclease domain was inactivated with the D450A mutation). ZFNickases were indeed active, resulting in ~250 ng/mL hF.IX 4 weeks post injection (Figure 1). Interestingly, we could not detect hF.IX in mice treated with ZFNickase and no-homology donor (LOD: 15ng/mL). To rule out the possibility that this was simply due to the lower efficacy of ZFNickases compared to ZFNs, we increased the ZFNickase dose 4 fold. Four weeks post treatment, we observed substantial levels of hF.IX in mice treated with homologous donor (2041 ± 269 ng/mL) and were again unable to detect hF.IX in mice treated with the non-homologous donor (n=10 and 7, respectively). These data point to homology directed repair as the primary mechanism of protein production for genome editing in neonatal mouse liver, and suggest improvements in both efficacy and specificity can be made through deeper understanding of the molecular requirements of this approach. [Display omitted] DisclosuresAnguela:Spark Therapeutics, Inc.: Employment, Equity Ownership, Patents & Royalties. Doyon:Sangamo BioSciences: Employment. Wechsler:Sangamo BioSciences: Employment. Paschon:Sangamo BioSciences: Employment. Davidson:Spark Therapeutics: Consultancy. Gregory:Sangamo BioSciences: Employment. Holmes:Sangamo BioSciences: Employment. High:Spark Therapeutics: Employment, Equity Ownership, Patents & Royalties.

Yeast Golden Gate (yGG) for the Efficient Assembly of S. cerevisiae Transcription Units.

We have adapted the Golden Gate DNA assembly method to the assembly of transcription units (TUs) for the yeast Saccharomyces cerevisiae, in a method we call yeast Golden Gate (yGG). yGG allows for the easy assembly of TUs consisting of promoters (PRO), coding sequences (CDS), and terminators (TER). Carefully designed overhangs exposed by digestion with a type IIS restriction enzyme enable virtually seamless assembly of TUs that, in principle, contain all of the information necessary to express a gene of interest in yeast. We also describe a versatile set of yGG acceptor vectors to be used for TU assembly. These vectors can be used for low or high copy expression of assembled TUs or integration into carefully selected innocuous genomic loci. yGG provides synthetic biologists and yeast geneticists with an efficient new means by which to engineer S. cerevisiae.

BioSpotlight Citations

BioSpotlight Citations

In Silico Identification and in Vitro Validation of Potential Cholestatic Compounds through 3D Ligand-Based Pharmacophore Modeling of BSEP Inhibitors

Drug-induced cholestasis is a frequently observed side effect of drugs and is often caused by an unexpected interaction with the bile salt export pump (BSEP/ABCB11). BSEP is the key membrane transporter responsible for the transport of bile acids from hepatocytes into bile. Here, we developed a pharmacophore model that describes the molecular features of compounds associated with BSEP inhibitory activity. To generate input and validation data sets, in vitro experiments with membrane vesicles overexpressing human BSEP were used to assess the effect of compounds (50 μM) on BSEP-mediated (3)H-taurocholic acid transport. The model contains two hydrogen bond acceptor/anionic features, two hydrogen bond acceptor vector features, four hydrophobic/aromatic features, and exclusion volumes. The pharmacophore was validated against a set of 59 compounds, including registered drugs. The model recognized 9 out of 12 inhibitors (75%), which could not be identified based on general parameters, such as molecular weight or SlogP, alone. Finally, the model was used to screen a virtual compound database. A number of compounds found via virtual screening were tested and displayed statistically significant BSEP inhibition, ranging from 13 ± 1% to 67 ± 7% of control (P < 0.05). In conclusion, we developed and validated a pharmacophore model that describes molecular features found in BSEP inhibitors. The model may be used as an in silico screening tool to identify potentially harmful drug candidates at an early stage in drug development.

ELOVL4 protein preferentially elongates 20:5n3 to very long chain PUFAs over 20:4n6 and 22:6n3

We hypothesized that reduction/loss of very long chain PUFAs (VLC-PUFAs) due to mutations in the ELOngase of very long chain fatty acid-4 (ELOVL4) protein contributes to retinal degeneration in autosomal dominant Stargardt-like macular dystrophy (STGD3) and age-related macular degeneration; hence, increasing VLC-PUFA in the retina of these patients could provide some therapeutic benefits. Thus, we tested the efficiency of elongation of C20-C22 PUFA by the ELOVL4 protein to determine which substrates are the best precursors for biosynthesis of VLC-PUFA. The ELOVL4 protein was expressed in pheochromocytoma cells, while green fluorescent protein-expressing and nontransduced cells served as controls. The cells were treated with 20:5n3, 22:6n3, and 20:4n6, either individually or in equal combinations. Both transduced and control cells internalized and elongated the supplemented FAs to C22-C26 precursors. Only ELOVL4-expressing cells synthesized C28-C38 VLC-PUFA from these precursors. In general, 20:5n3 was more efficiently elongated to VLC-PUFA in the ELOVL4-expressing cells, regardless of whether it was in combination with 22:6n3 or with 20:4n6. In each FA treatment group, C34 and C36 VLC-PUFAs were the predominant VLC-PUFAs in the ELOVL4-expressing cells. In summary, 20:5n3, followed by 20:4n6, seems to be the best precursor for boosting the synthesis of VLC-PUFA by ELOVL4 protein.

Citations

Citations

Development and Characterization of High Affinity Leptins and Leptin Antagonists

Leptin is a pleiotropic hormone acting both centrally and peripherally. It participates in a variety of biological processes, including energy metabolism, reproduction, and modulation of the immune response. So far, structural elements affecting leptin binding to its receptor remain unknown. We employed random mutagenesis of leptin, followed by selection of high affinity mutants by yeast surface display and discovered that replacing residue Asp-23 with a non-negatively charged amino acid leads to dramatically enhanced affinity of leptin for its soluble receptor. Rational mutagenesis of Asp-23 revealed the D23L substitution to be most effective. Coupling the Asp-23 mutation with alanine mutagenesis of three amino acids (L39A/D40A/F41A) previously reported to convert leptin into antagonist resulted in potent antagonistic activity. These novel superactive mouse and human leptin antagonists (D23L/L39A/D40A/F41A), termed SMLA and SHLA, respectively, exhibited over 60-fold increased binding to leptin receptor and 14-fold higher antagonistic activity in vitro relative to the L39A/D40A/F41A mutants. To prolong and enhance in vivo activity, SMLA and SHLA were monopegylated mainly at the N terminus. Administration of the pegylated SMLA to mice resulted in a remarkably rapid, significant, and reversible 27-fold more potent increase in body weight (as compared with pegylated mouse leptin antagonist), because of increased food consumption. Thus, recognition and mutagenesis of Asp-23 enabled construction of novel compounds that induce potent and reversible central and peripheral leptin deficiency. In addition to enhancing our understanding of leptin interactions with its receptor, these antagonists enable in vivo study of the role of leptin in metabolic and immune processes and hold potential for future therapeutic use in disease pathologies involving leptin.

Cre-lox univector acceptor vectors for functional screening in protoplasts: analysis of Arabidopsis donor cDNAs encoding ABSCISIC ACID INSENSITIVE1-like protein phosphatases.

The 14,200 available full length Arabidopsis thaliana cDNAs in the universal plasmid system (UPS) donor vector pUNI51 should be applied broadly and efficiently to leverage a "functional map-space" of homologous plant genes. We have engineered Cre-lox UPS host acceptor vectors (pCR701- 705) with N-terminal epitope tags in frame with the loxH site and downstream from the maize Ubiquitin promoter for use in transient protoplast expression assays and particle bombardment transformation of monocots. As an example of the utility of these vectors, we recombined them with several Arabidopsis cDNAs encoding Ser/Thr protein phosphatase type 2C (PP2Cs) known from genetic studies or predicted by hierarchical clustering meta-analysis to be involved in ABA and stress responses. Our functional results in Zea mays mesophyll protoplasts on ABA-inducible expression effects on the Late Embryogenesis Abundant promoter ProEm:GUS reporter were consistent with predictions and resulted in identification of novel activities of some PP2Cs. Deployment of these vectors can facilitate functional genomics and proteomics and identification of novel gene activities.

Golden Gate Shuffling: A One-Pot DNA Shuffling Method Based on Type IIs Restriction Enzymes

We have developed a protocol to assemble in one step and one tube at least nine separate DNA fragments together into an acceptor vector, with 90% of recombinant clones obtained containing the desired construct. This protocol is based on the use of type IIs restriction enzymes and is performed by simply subjecting a mix of 10 undigested input plasmids (nine insert plasmids and the acceptor vector) to a restriction-ligation and transforming the resulting mix in competent cells. The efficiency of this protocol allows generating libraries of recombinant genes by combining in one reaction several fragment sets prepared from different parental templates. As an example, we have applied this strategy for shuffling of trypsinogen from three parental templates (bovine cationic trypsinogen, bovine anionic trypsinogen and human cationic trypsinogen) each divided in 9 separate modules. We show that one round of shuffling using the 27 trypsinogen entry plasmids can easily produce the 19,683 different possible combinations in one single restriction-ligation and that expression screening of a subset of the library allows identification of variants that can lead to higher expression levels of trypsin activity. This protocol, that we call ‘Golden Gate shuffling’, is robust, simple and efficient, can be performed with templates that have no homology, and can be combined with other shuffling protocols in order to introduce any variation in any part of a given gene.

A Hybrid Vector System Expands Adeno-associated Viral Vector Packaging Capacity in a Transgene-independent Manner

The trans-splicing (ts) and overlapping (ov) vectors expand the packaging capacity of adeno-associated virus (AAV). But their application depends on the inherent properties of the target gene. The ts vectors require an optimal gene-splitting site and the ov vectors require a highly recombinogenic domain. In order to overcome these limitations, we developed a hybrid dual (hd) vector system. In the hd vectors, we inserted a highly recombinogenic alkaline phosphatase (AP) sequence in the ts vectors to allow for transgene-independent reconstitution through homologous recombination of the AP sequences. We first tested the hybrid system with the LacZ gene. Both in the cell line (in vitro) and in the mouse muscle (in vivo), the hd vectors significantly outperformed the ts and ov vectors. In muscle, the transduction efficiency of the hybrid vectors reached 80% of that from the single intact vector. Southern blot confirmed AP sequence-mediated transgene reconstitution. In order to validate the hybrid system, we split the 6 kilobase (kb) mini-dystrophin gene at the exon 55/56 junction, a predicted poor site for the ts approach. In dystrophic mdx mouse muscle, the hd vectors yielded 5.6-fold higher transduction than the ts vectors did. Taken together, these data suggest that the hybrid system efficiently expresses large therapeutic genes that are poor candidates for the ts and ov approaches.

Generation of retroviruses for the overexpression of cytosolic and mitochondrial glutathione reductase in macrophages in vivo

Retroviral gene transfer and bone marrow transplantation has been used by many investigators to study the role of macrophage proteins in different mouse models of human disease. While this approach is faster and less expensive than generating transgenic mice with macrophage-specific promoters and applicable to a wider array of mouse models, it has been hampered by two major drawbacks: labor-intensive cloning procedures involved in generating retroviral vectors for each gene of interest and low viral titers. Here we describe the construction of a MSCV-based retroviral vector that can serve as an acceptor vector for commercially available Cre-lox-compatible donor vectors. Using this new retroviral vector in combination with a FACS approach to enhance viral titers, we generated high-titer retroviruses carrying either EGFP-tagged cytosolic or EGFP-tagged mitochondria-targeted glutathione reductase. We show that the introduction of these constructs via retroviral gene transfer and bone marrow transplantation into atherosclerosis-prone LDL receptor-null mice results in the long-term increase in macrophage glutathione reductase activity.

Functional Characterization of PmHS1, a Pasteurella multocida Heparosan Synthase

Heparosan synthase 1 (PmHS1) from Pasteurella multocida Type D is a dual action glycosyltransferase enzyme that transfers monosaccharide units from uridine diphospho (UDP) sugar precursors to form the polysaccharide heparosan (N-acetylheparosan), which is composed of alternating (-alpha4-GlcNAc-beta1,4-GlcUA-1-) repeats. We have used molecular genetic means to remove regions nonessential for catalytic activity from the amino- and the carboxyl-terminal regions as well as characterized the functional regions involved in GlcUA-transferase activity and in GlcNAc-transferase activity. Mutation of either one of the two regions containing aspartate-X-aspartate (DXD) residue-containing motifs resulted in complete or substantial loss of heparosan polymerizing activity. However, certain mutant proteins retained only GlcUA-transferase activity while some constructs possessed only GlcNAc-transferase activity. Therefore, it appears that the PmHS1 polypeptide is composed of two types of glycosyltransferases in a single polypeptide as was found for the Pasteurella multocida Type A PmHAS, the hyaluronan synthase that makes the alternating (-beta3-GlcNAc-beta1,4-GlcUA-1-) polymer. However, there is low amino acid similarity between the PmHAS and PmHS1 enzymes, and the relative placement of the GlcUA-transferase and GlcNAc-transferase domains within the two polypeptides is reversed. Even though the monosaccharide compositions of hyaluronan and heparosan are identical, such differences in the sequences of the catalysts are expected because the PmHAS employs only inverting sugar transfer mechanisms whereas PmHS1 requires both retaining and inverting mechanisms.

Modification of the Creator recombination system for proteomics applications – improved expression by addition of splice sites

BackgroundRecombinational systems have been developed to rapidly shuttle Open Reading Frames (ORFs) into multiple expression vectors in order to analyze the large number of cDNAs available in the post-genomic era. In the Creator system, an ORF introduced into a donor vector can be transferred with Cre recombinase to a library of acceptor vectors optimized for different applications. Usability of the Creator system is impacted by the ability to easily manipulate DNA, the number of acceptor vectors for downstream applications, and the level of protein expression from Creator vectors.ResultsTo date, we have developed over 20 novel acceptor vectors that employ a variety of promoters and epitope tags commonly employed for proteomics applications and gene function analysis. We also made several enhancements to the donor vectors including addition of different multiple cloning sites to allow shuttling from pre-existing vectors and introduction of the lacZ alpha reporter gene to allow for selection. Importantly, in order to ameliorate any effects on protein expression of the loxP site between a 5' tag and ORF, we introduced a splicing event into our expression vectors. The message produced from the resulting 'Creator Splice' vector undergoes splicing in mammalian systems to remove the loxP site. Upon analysis of our Creator Splice constructs, we discovered that protein expression levels were also significantly increased.ConclusionThe development of new donor and acceptor vectors has increased versatility during the cloning process and made this system compatible with a wider variety of downstream applications. The modifications introduced in our Creator Splice system were designed to remove extraneous sequences due to recombination but also aided in downstream analysis by increasing protein expression levels. As a result, we can now employ epitope tags that are detected less efficiently and reduce our assay scale to allow for higher throughput. The Creator Splice system appears to be an extremely useful tool for proteomics.

821. Successful Adaptation of the RCAS-TVA Gene Transfer System for Use in a Bovine Mammary Epithelial Cell Culture Model

The RCAS-TVA gene transfer system is based on a recombinant avian leucosis virus which is produced by the replication-competent avian splice acceptor vector, RCAS, and requires the presence of the avian subtype A receptor, TVA, for infection. Mammalian cells can be infected, however this requires these cells to be engineered to express the TVA receptor. Significant biosafety features include the virus being replication competent only in avian cells as there is inefficient viral assembly in mammalian cells even in those modified for infection.