A synthetic, closed-looped gene circuit for the autonomous regulation of RUNX2 activity during chondrogenesis.

The transcription factor RUNX2 is a key regulator of chondrocyte phenotype during development, making it an ideal target for prevention of undesirable chondrocyte maturation in cartilage tissue-engineering strategies. Here, we engineered an autoregulatory gene circuit (cisCXp-shRunx2) that negatively controls RUNX2 activity in chondrogenic cells via RNA interference initiated by a tunable synthetic Col10a1-like promoter (cisCXp). The cisCXp-shRunx2 gene circuit is designed based on the observation that induced RUNX2 silencing after early chondrogenesis enhances the accumulation of cartilaginous matrix in ATDC5 cells. We show that the cisCXp-shRunx2 initiates RNAi of RUNX2 in maturing chondrocytes in response to the increasing intracellular RUNX2 activity without interfering with early chondrogenesis. The induced loss of RUNX2 activity in turn negatively regulates the gene circuit itself. Moreover, the efficacy of RUNX2 suppression from cisCXp-shRunx2 can be controlled by modifying the sensitivity of cisCXp promoter. Finally, we show the efficacy of inhibiting RUNX2 in preventing matrix loss in human mesenchymal stem cell-derived (hMSC-derived) cartilage under conditions that induce chondrocyte hypertrophic differentiation, including inflammation. Overall, our results demonstrated that the negative modulation of RUNX2 activity with our autoregulatory gene circuit enhanced matrix synthesis and resisted ECM degradation by reprogrammed MSC-derived chondrocytes in response to the microenvironment of the degenerative joint.

Effects of intracardiac delivery of aldehyde dehydrogenase 2 gene in myocardial salvage.

Intrinsic activity of aldehyde dehydrogenase (ALDH)2, a cardiac mitochondrial enzyme, is vital in detoxifying 4-hydroxy-2-nonenal (4HNE) like cellular reactive carbonyl species (RCS) and thereby conferring cardiac protection against pathological stress. It was also known that a single point mutation (E487K) in ALDH2 (prevalent in East Asians) known as ALDH2*2 reduces its activity intrinsically and was associated with increased cardiovascular diseases. We and others have shown that ALDH2 activity is reduced in several pathologies in WT animals as well. Thus, exogenous augmentation of ALDH2 activity is a good strategy to protect the myocardium from pathologies. In this study, we will test the efficacy of intracardiac injections of the ALDH2 gene in mice. We injected both wild type (WT) and ALDH2*2 knock-in mutant mice with ALDH2 constructs, AAv9-cTNT-hALDH2-HA tag-P2A-eGFP or their control constructs, AAv9-cTNT-eGFP. We found that intracardiac ALDH2 gene transfer increased myocardial levels of ALDH2 compared to GFP alone after 1 and 3 weeks. When we subjected the hearts of these mice to 30 min global ischemia and 90 min reperfusion (I-R) using the Langendorff perfusion system, we found reduced infarct size in the hearts of mice with ALDH2 gene vs GFP alone. A single time injection has shown increased myocardial ALDH2 activity for at least 3 weeks and reduced myocardial 4HNE adducts and infarct size along with increased contractile function of the hearts while subjected to I-R. Thus, ALDH2 overexpression protected the myocardium from I-R injury by reducing 4HNE protein adducts implicating increased 4HNE detoxification by ALDH2. In conclusion, intracardiac ALDH2 gene transfer is an effective strategy to protect the myocardium from pathological insults.

A neurodegeneration gene, WDR45, links impaired ferritinophagy to iron accumulation.

Neurodegeneration with brain iron accumulation (NBIA) is a clinically and genetically heterogeneous group of neurodegenerative diseases characterized by the abnormal accumulation of brain iron and the progressive degeneration of the nervous system. One of the recently identified subtypes of NBIA is ß-propeller protein-associated neurodegeneration (BPAN). BPAN is caused by de novo mutations in the WDR45/WIPI4 (WD repeat domain 45) gene. WDR45 is one of the four mammalian homologs of yeast Atg18, a regulator of autophagy. WDR45 deficiency in BPAN patients and animal models may result in defects in autophagic flux. However, how WDR45 deficiency leads to brain iron overload remains unclear. To elucidate the role of WDR45, we generated a WDR45-knockout (KO) SH-SY5Y neuroblastoma cell line using CRISPR-Cas9-mediated genome editing. Using these cells, we demonstrated that the non-TF (transferrin)-bound iron pathway dominantly mediated the accumulation of iron. Moreover, the loss of WDR45 led to defects in ferritinophagy, a form of autophagy that degrades the iron storage protein ferritin. We showed that impaired ferritinophagy contributes to iron accumulation in WDR45-KO cells. Iron accumulation was also detected in the mitochondria, which was accompanied by impaired mitochondrial respiration, elevated reactive oxygen species, and increased cell death. Thus, our study links WDR45 to specific iron acquisition pathways and ferritinophagy. Cover Image for this issue: https://doi.org/10.1111/jnc.15388.

Discovery and characterization of high-affinity, potent SARS-CoV-2 neutralizing antibodies via single B cell screening.

Monoclonal antibodies that target SARS-CoV-2 with high affinity are valuable for a wide range of biomedical applications involving novel coronavirus disease (COVID-19) diagnosis, treatment, and prophylactic intervention. Strategies for the rapid and reliable isolation of these antibodies, especially potent neutralizing antibodies, are critical toward improved COVID-19 response and informed future response to emergent infectious diseases. In this study, single B cell screening was used to interrogate antibody repertoires of immunized mice and isolate antigen-specific IgG1+ memory B cells. Using these methods, high-affinity, potent neutralizing antibodies were identified that target the receptor-binding domain of SARS-CoV-2. Further engineering of the identified molecules to increase valency resulted in enhanced neutralizing activity. Mechanistic investigation revealed that these antibodies compete with ACE2 for binding to the receptor-binding domain of SARS-CoV-2. These antibodies may warrant further development for urgent COVID-19 applications. Overall, these results highlight the potential of single B cell screening for the rapid and reliable identification of high-affinity, potent neutralizing antibodies for infectious disease applications.

Engineered Multivalent Nanobodies Potently and Broadly Neutralize SARS-CoV-2 Variants.

The COVID-19 pandemic continues to be a severe threat to human health, especially due to current and emerging SARS-CoV-2 variants with potential to escape humoral immunity developed after vaccination or infection. The development of broadly neutralizing antibodies that engage evolutionarily conserved epitopes on coronavirus spike proteins represents a promising strategy to improve therapy and prophylaxis against SARS-CoV-2 and variants thereof. Herein, a facile multivalent engineering approach is employed to achieve large synergistic improvements in the neutralizing activity of a SARS-CoV-2 cross-reactive nanobody (VHH-72) initially generated against SARS-CoV. This synergy is epitope specific and is not observed for a second high-affinity nanobody against a non-conserved epitope in the receptor-binding domain. Importantly, a hexavalent VHH-72 nanobody retains binding to spike proteins from multiple highly transmissible SARS-CoV-2 variants (B.1.1.7 and B.1.351) and potently neutralizes them. Multivalent VHH-72 nanobodies also display drug-like biophysical properties, including high stability, high solubility, and low levels of non-specific binding. The unique neutralizing and biophysical properties of VHH-72 multivalent nanobodies make them attractive as therapeutics against SARS-CoV-2 variants.

Directed evolution of potent neutralizing nanobodies against SARS-CoV-2 using CDR-swapping mutagenesis.

There is widespread interest in facile methods for generating potent neutralizing antibodies, nanobodies, and other affinity proteins against SARS-CoV-2 and related viruses to address current and future pandemics. While isolating antibodies from animals and humans are proven approaches, these methods are limited to the affinities, specificities, and functional activities of antibodies generated by the immune system. Here we report a surprisingly simple directed evolution method for generating nanobodies with high affinities and neutralization activities against SARS-CoV-2. We demonstrate that complementarity-determining region swapping between low-affinity lead nanobodies, which we discovered unintentionally but find is simple to implement systematically, results in matured nanobodies with unusually large increases in affinity. Importantly, the matured nanobodies potently neutralize both SARS-CoV-2 pseudovirus and live virus, and possess drug-like biophysical properties. We expect that our methods will improve in vitro nanobody discovery and accelerate the generation of potent neutralizing nanobodies against diverse coronaviruses.

IgV somatic mutation of human anti-SARS-CoV-2 monoclonal antibodies governs neutralization and breadth of reactivity.

Abs that neutralize SARS-CoV-2 are thought to provide the most immediate and effective treatment for those severely afflicted by this virus. Because coronavirus potentially diversifies by mutation, broadly neutralizing Abs are especially sought. Here, we report a possibly novel approach to rapid generation of potent broadly neutralizing human anti-SARS-CoV-2 Abs. We isolated SARS-CoV-2 spike protein-specific memory B cells by panning from the blood of convalescent subjects after infection with SARS-CoV-2 and sequenced and expressed Ig genes from individual B cells as human mAbs. All of 43 human mAbs generated in this way neutralized SARS-CoV-2. Eighteen of the forty-three human mAbs exhibited half-maximal inhibitory concentrations (IC50) of 6.7 × 10-12 M to 6.7 × 10-15 M for spike-pseudotyped virus. Seven of the human mAbs also neutralized (with IC50 < 6.7 × 10-12 M) viruses pseudotyped with mutant spike proteins (including receptor-binding domain mutants and the S1 C-terminal D614G mutant). Neutralization of the Wuhan Hu-1 founder strain and of some variants decreased when coding sequences were reverted to germline, suggesting that potency of neutralization was acquired by somatic hypermutation and selection of B cells. These results indicate that infection with SARS-CoV-2 evokes high-affinity B cell responses, some products of which are broadly neutralizing and others highly strain specific. We also identify variants that would potentially resist immunity evoked by infection with the Wuhan Hu-1 founder strain or by vaccines developed with products of that strain, suggesting evolutionary courses that SARS-CoV-2 could take.

CD6 is a target for cancer immunotherapy.

Limitations of checkpoint inhibitor cancer immunotherapy include induction of autoimmune syndromes and resistance of many cancers. Since CD318, a novel CD6 ligand, is associated with the aggressiveness and metastatic potential of human cancers, we tested the effect of an anti-CD6 monoclonal antibody, UMCD6, on killing of cancer cells by human lymphocytes. UMCD6 augmented killing of breast, lung, and prostate cancer cells through direct effects on both CD8+ T cells and NK cells, increasing cancer cell death and lowering cancer cell survival in vitro more robustly than monoclonal antibody checkpoint inhibitors that interrupt the programmed cell death 1 (PD-1)/PD-1 ligand 1 (PD-L1) axis. UMCD6 also augmented in vivo killing by human peripheral blood lymphocytes of a human breast cancer line xenotransplanted into immunodeficient mice. Mechanistically, UMCD6 upregulated the expression of the activating receptor NKG2D and downregulated expression of the inhibitory receptor NKG2A on both NK cells and CD8+ T cells, with concurrent increases in perforin and granzyme B production. The combined capability of an anti-CD6 monoclonal antibody to control autoimmunity through effects on CD4+ lymphocyte differentiation while enhancing killing of cancer cells through distinct effects on CD8+ and NK cells opens a potential new approach to cancer immunotherapy that would suppress rather than instigate autoimmunity.

Real time visualization of cancer cell death, survival and proliferation using fluorochrome-transfected cells in an IncuCyte® imaging system.

Cancer immunotherapy is a rapidly advancing and viable approach to treating cancer along with more traditional forms of therapy. Real-time cell analysis technologies that examine the dynamic interactions between cancer cells and the cells of the immune system are becoming more important for assessment of novel therapeutics. In this report, we use the IncuCyte® imaging system to study the killing potential of various immune cells on cancer cell lines. The IncuCyte® system tracks living cells, labeled by a red fluorescent protein, and cell death, as indicated by the caspase-3/7 reagent, which generates a green fluorescent signal upon activation of apoptotic pathways. Despite the power of this approach, obtaining commercially fluorescent cancer cell lines is expensive and limited in the range of cell lines that are available. To overcome this barrier, we developed an inexpensive method using a lentiviral construct expressing nuclear localized mKate2 red fluorescent protein to stably label cancer cells. We demonstrate that this method is effective in labeling a wide variety of cell lines, allowing for analyses of different cancers as well as different cell lines of the same type of cancer.

Principles of Genetic Engineering.

Genetic engineering is the use of molecular biology technology to modify DNA sequence(s) in genomes, using a variety of approaches. For example, homologous recombination can be used to target specific sequences in mouse embryonic stem (ES) cell genomes or other cultured cells, but it is cumbersome, poorly efficient, and relies on drug positive/negative selection in cell culture for success. Other routinely applied methods include random integration of DNA after direct transfection (microinjection), transposon-mediated DNA insertion, or DNA insertion mediated by viral vectors for the production of transgenic mice and rats. Random integration of DNA occurs more frequently than homologous recombination, but has numerous drawbacks, despite its efficiency. The most elegant and effective method is technology based on guided endonucleases, because these can target specific DNA sequences. Since the advent of clustered regularly interspaced short palindromic repeats or CRISPR/Cas9 technology, endonuclease-mediated gene targeting has become the most widely applied method to engineer genomes, supplanting the use of zinc finger nucleases, transcription activator-like effector nucleases, and meganucleases. Future improvements in CRISPR/Cas9 gene editing may be achieved by increasing the efficiency of homology-directed repair. Here, we describe principles of genetic engineering and detail: (1) how common elements of current technologies include the need for a chromosome break to occur, (2) the use of specific and sensitive genotyping assays to detect altered genomes, and (3) delivery modalities that impact characterization of gene modifications. In summary, while some principles of genetic engineering remain steadfast, others change as technologies are ever-evolving and continue to revolutionize research in many fields.

Optimization of overlap extension PCR for efficient transgene construction.

PCR is a powerful tool for generating specific fragments of DNA that can be used to create gene variations or tagged expression constructs. Overlap extension PCR is a valuable technique that is commonly used for cloning large complex fragments, making edits to cloned genes or fusing two gene elements together. After difficulties in utilizing this technique following existing methods, we developed an optimized protocol. To accomplish this, three significant changes were made; 1) touchdown PCR cycling parameters were used to eliminate the need for optimizing PCR cycling conditions, 2) the high-fidelity, high-processivity Q5 DNA polymerase was used to improve full-length amplification quality, and 3) a reduced amount of primer in the final PCR amplification step decreased non-specific amplimers. This modified protocol results in consistent generation of gene fusion products, with little to no background and enhanced efficiency of the transgene construction process.

Development and validation of a reporter cell line for rapid AAV quality control assessment.

Adenovirus-associated virus is a powerful vector system for transducing cells in vivo. It is widely used in animal systems due to high transduction efficiency of non-dividing cells with more than a dozen serotypes that have preferential tissue tropism. The viral genome remains episomal in the nucleus but maintains sustained expression in terminally differentiated cells for several weeks to months. Despite the popularity of recombinant AAV (rAAV) vectors, quality control testing of the virus after production is largely limited to physical characteristics such as viral genomes/ml determinations and silver staining acrylamide gels to determine purity. Functional testing, in vivo, is not practical due to high cost and restricted access of animal care and long duration of the assay (2-3 weeks). Some functional testing can be accomplished in cultured cells such as HEK293 cells, but HEK293 cells limit the types of rAAV constructs that can be tested. Many rAAV constructs are designed to study neurons in the brain with neural-specific promoters and many are floxed with loxp sites to be "activated" only in Cre-expressing neurons in transgenic animals. To develop a reporter cell line for rapid rAAV quality control assessment of these neural-specific, floxed rAAV constructs, we used the lentiviral system to stably express Cre recombinase in the SH-SY5Y neuroblastoma cell line. •A simple and economic method to evaluate recombinant AAV in vitro.•Allows functional validation of rAAV across a wide range of serotypes and promoters.•Allows functional validation of Cre-dependent rAAV constructs.

Citrullinated Inhibitor of DNA Binding 1 Is a Novel Autoantigen in Rheumatoid Arthritis.

OBJECTIVE: To explore the intrinsic role of inhibitor of DNA binding 1 (ID-1) in rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLS) and to investigate whether ID-1 is citrullinated and autoantigenic in RA. METHODS: RA patient serum ID-1 levels were measured before and after infliximab treatment. RA FLS were transfected with a clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 construct targeting ID-1 to examine the effects of ID-1 deletion. RA synovial fluid (SF) and homogenized synovial tissue (ST) were immunoprecipitated for ID-1 and measured for citrullinated residues using an enzyme-linked immunosorbent assay and Western blotting. Liquid chromatography tandem mass spectrometry (LC-MS/MS) was performed on in vitro-citrullinated recombinant human ID-1 (cit-ID-1) to localize the sites of citrullination. Normal and RA sera and SF were analyzed by immunodot blotting for anti-citrullinated protein antibodies (ACPAs) to cit-ID-1. RESULTS: RA patient serum ID-1 levels positively correlated with several disease parameters and were reduced after infliximab treatment. RA FLS displayed reduced growth and a robust increase in interleukin-6 (IL-6) and IL-8 production upon deletion of ID-1. ID-1 immunodepletion significantly reduced the levels of citrullinated residues in RA SF, and citrullinated ID-1 was detected in homogenized RA ST (n = 5 samples; P < 0.05). Immunodot blot analyses revealed ACPAs to cit-ID-1 but not to native ID-1, in RA peripheral blood (PB) sera (n = 30 samples; P < 0.001) and SF (n = 18 samples; P < 0.05) but not in normal PB sera. Following analyses of LC-MS/MS results for citrullination sites and corresponding reactivity in immunodot assays, we determined the critical arginines in ID-1 for autoantigenicity: R33, R52, and R121. CONCLUSION: Novel roles of ID-1 in RA include regulation of FLS proliferation and cytokine secretion as well as autoantigenicity following citrullination.

A leptin-regulated circuit controls glucose mobilization during noxious stimuli.

Adipocytes secrete the hormone leptin to signal the sufficiency of energy stores. Reductions in circulating leptin concentrations reflect a negative energy balance, which augments sympathetic nervous system (SNS) activation in response to metabolically demanding emergencies. This process ensures adequate glucose mobilization despite low energy stores. We report that leptin receptor-expressing neurons (LepRb neurons) in the periaqueductal gray (PAG), the largest population of LepRb neurons in the brain stem, mediate this process. Application of noxious stimuli, which often signal the need to mobilize glucose to support an appropriate response, activated PAG LepRb neurons, which project to and activate parabrachial nucleus (PBN) neurons that control SNS activation and glucose mobilization. Furthermore, activating PAG LepRb neurons increased SNS activity and blood glucose concentrations, while ablating LepRb in PAG neurons augmented glucose mobilization in response to noxious stimuli. Thus, decreased leptin action on PAG LepRb neurons augments the autonomic response to noxious stimuli, ensuring sufficient glucose mobilization during periods of acute demand in the face of diminished energy stores.

Increased Activation of the TRESK K+ Mediates Vago-Vagal Reflex Malfunction in Diabetic Rats.

BACKGROUND & AIMS: Patients with diabetes have defects in the vagal afferent pathway that result in abnormal gastrointestinal function. We investigated whether selective increased activation of the 2-pore domain potassium channel TRESK (2-pore-domain weak inward-rectifying potassium channel-related spinal cord potassium channel) contributes to nodose ganglia (NG) malfunction, disrupting gastrointestinal function in diabetic rats. METHODS: We conducted whole-cell current-clamp and single-unit recordings in NG neurons from diabetes-prone BioBreeding/Worcester rats and streptozotocin-induced diabetic (STZ-D) rats and compared them with control rats. NG neurons in rats or cultured NG neurons were exposed to pharmacologic antagonists and/or transfected with short hairpin or small interfering RNAs that reduced expression of TRESK. We then made electrophysiologic recordings and studied gastrointestinal functions. RESULTS: We observed reduced input resistance, hyperpolarized membrane potential, and increased current threshold to elicit action potentiation in NG neurons of STZ-D rats compared with controls. NG neuron excitability was similarly altered in diabetes-prone rats. In vivo single-unit NG neuronal discharges in response to 30 and 60 pmol cholecystokinin octapeptide were significantly lower in STZ-D rats compared with controls. Reducing expression of the TRESK K+ channel restored NG excitability in vitro and in vivo, as well as cholecystokinin 8-stimulated secretion of pancreatic enzymes and secretin-induced gastrointestinal motility, which are mediated by vago-vagal reflexes. These abnormalities resulted from increased intracellular Ca2+ in the NG, activating calcineurin, which, in turn, bound to an nuclear factor of activated T cell-like docking site on the TRESK protein, resulting in neuronal membrane hyperpolarization. CONCLUSIONS: In 2 rate models of diabetes, we found that activation of the TRESK K+ channel reduced NG excitability and disrupted gastrointestinal functions.

Expression and function of aminopeptidase N/CD13 produced by fibroblast-like synoviocytes in rheumatoid arthritis: role of CD13 in chemotaxis of cytokine-activated T cells independent of enzymatic activity.

OBJECTIVE: Aminopeptidase N/CD13 (EC 3.4.11.2) is a metalloproteinase expressed by fibroblast-like synoviocytes (FLS). It has been suggested that CD13 can act chemotactically for T cells in rheumatoid arthritis (RA). We undertook this study to measure CD13 in vivo and in vitro in RA samples and to determine whether CD13 could play a role in the homing of T cells to the RA joint. METHODS: Interleukin-17-treated FLS were used to immunize mice, from which a novel anti-human CD13 monoclonal antibody (mAb), 591.1D7.34, was developed. The mAb 591.1D7.34 and a second anti-CD13 mAb, WM15, were used to develop a novel enzyme-linked immunosorbent assay (ELISA) for CD13, and CD13 enzymatic activity was measured in parallel. Chemotaxis of cytokine-activated T cells was measured by a chemotaxis-under-agarose assay. RESULTS: We detected substantial amounts of CD13 in synovial fluid (SF), sera, FLS lysates, and culture supernatants by ELISA, with a significant increase in CD13 in RA SF when compared to osteoarthritis SF. CD13 accounted for most but not all of the CD13-like enzymatic activity in SF. Recombinant human CD13 was chemotactic for cytokine-activated T cells through a G protein-coupled receptor and contributed to the chemotactic properties of SF independently of enzymatic activity. CONCLUSION: CD13 is released from FLS into culture supernatants and is found in SF. CD13 induces chemotaxis of cytokine-activated T cells, a T cell population similar to that found in RA synovium. These data suggest that CD13 could play an important role as a T cell chemoattractant, in a positive feedback loop that contributes to RA synovitis.

GATA-3 expression identifies a high-risk subset of PTCL, NOS with distinct molecular and clinical features.

The cell of origin and the tumor microenvironment's role remain elusive for the most common peripheral T-cell lymphomas (PTCLs). As macrophages promote the growth and survival of malignant T cells and are abundant constituents of the tumor microenvironment, their functional polarization was examined in T-cell lymphoproliferative disorders. Cytokines that are abundant within the tumor microenvironment, particularly interleukin (IL)-10, were observed to promote alternative macrophage polarization. Macrophage polarization was signal transducer and activator of transcription 3 dependent and was impaired by the Janus kinase inhibitor ruxolitinib. In conventional T cells, the production of T helper (Th)2-associated cytokines and IL-10, both of which promote alternative macrophage polarization, is regulated by the T-cell transcription factor GATA-binding protein 3 (GATA-3). Therefore, its role in the T-cell lymphomas was examined. GATA-3 expression was observed in 45% of PTCLs, not otherwise specified (PTCL, NOS) and was associated with distinct molecular features, including the production of Th2-associated cytokines. In addition, GATA-3 expression identified a subset of PTCL, NOS with distinct clinical features, including inferior progression-free and overall survival. Collectively, these data suggest that further understanding the cell of origin and lymphocyte ontogeny among the T-cell lymphomas may improve our understanding of the tumor microenvironment's pathogenic role in these aggressive lymphomas.

Bile acid-oligopeptide conjugates interact with DNA and facilitate transfection.

Bile acids conjugated to oligoarginine-containing peptides (BACs) form complexes with DNA based on the electrostatic interactions between negatively charged phosphate groups of the nucleic acid and the positively charged side chain guanidinium groups of the oligoarginine in the BACs. Charge neutralization of both components and subsequent increases of the net positive charge of the complex combined with the water-soluble lipophilic nature of the bile acid results in changes in the physicochemistry and biological properties of the complexes. We have examined the relationship of a series of 13 BACs on their interaction with circular plasmid DNA (pDNA). The formation of soluble, low-density and insoluble, high-density complexes was analyzed using several methods. The formation of high-density complexes was dependent on the DNA concentration, and was enhanced by increasing the BAC to pDNA charge ratio. Several of the BAC:pDNA complexes demonstrated exclusion of the DNA-intercalator Hoechst 33258 from pDNA, and were also protected from DNase activity. Several BAC conjugates interacted with pDNA to form nanometer-sized particles suitable for cell transfection in vitro. Five of the 13 BACs were transfection competent as single agents, and 11 of the 13 BACs showed enhancement of transfection in combination with DOPE containing liposomes or silica nanoparticles.

Development and characterization of a canine oral mucosa equivalent in a serum-free environment.

The objectives of this study were to develop a serum-free system for culturing canine oral keratinocytes, the construction and characterization of a canine ex vivo produced oral mucosa equivalent (EVPOME), and transduction green fluorescent protein (GFP) into keratinocytes as a post-grafting tracking marker. Dissociated canine buccal mucosa keratinocytes were cultured in a chemically defined serum-free medium, Epilife trade mark. First-passage keratinocytes were transfected with the GFP gene using a lentiviral vector, sorted by flow cytometer and seeded onto a dermal equivalent, AlloDerm(R) to form EVPOMEs. The EVPOME was characterized by histology and immunohistochemistry, for p63, Ki-67, and involucrin. Laser confocal microscopy was used to locate GFP-transfected keratinocytes within the EVPOME. Cultured canine oral keratinocytes grew rapidly over the first three passages and then the proliferative rate decreased. The canine EVPOME formed a well-stratified epithelial layer. The majority of p63 and Ki-67 immunopositive cells were located in the basal layer whereas cytoplasmic involucrin expression was seen in the suprabasal layers, similar to native canine buccal mucosa. Under laser confocal microscopy, significant green fluorescence was observed throughout the EVPOME. In conclusion, canine EVPOMEs were successfully fabricated in a defined serum-free system with similar characteristics to native buccal mucosa. GFP-transfected canine oral keratinocytes could be identified within the EVPOME.