Introduction Of Transgenes Research Articles

Engineered T cell therapies.

Alongside advancements in gene therapy for inherited immune disorders, the need for effective alternative therapeutic options for other conditions has resulted in an expansion in the field of research for T cell gene therapy. T cells are easily obtained and can be induced to divide robustly ex vivo, a characteristic that allows them to be highly permissible to viral vector-mediated introduction of transgenes. Pioneering clinical trials targeting cancers and infectious diseases have provided safety and feasibility data and important information about persistence of engineered cells in vivo. Here, we review clinical experiences with γ-retroviral and lentiviral vectors and consider the potential of integrating transposon-based vectors as well as specific genome editing with designer nucleases in engineered T cell therapies.

Ex-post analysis of landraces sympatric to a commercial variety in the center of origin of the potato failed to detect gene flow.

The possible introduction of genetically modified potato in the Andean region raises concerns about the unintentional introduction of transgenes into the native potato germplasm because it is perceived to convey negative impacts on biodiversity. We investigated this question by an ex-post analysis of existing landraces resulting from natural hybridization between an unknown landrace and the fertile commercial variety ‘Yungay’. This variety can be regarded as exotic because it was bred in part from the southern Chilean germplasm of Solanum tuberosum Group Chilotanum. We sampled the landrace germplasm of 1,771 leaf samples comprising more than 400 different landraces from three regions where ‘Yungay’ and landraces have coexisted for 15–25 years in the Peruvian Andes. Simple sequence repeat (SSR) markers were used to identify putative hybrids based on allele sharing with those of ‘Yungay’. The exclusion procedure was iterative, starting with the SSR markers with highest discriminating capacity based on allele frequency of the variety ‘Yungay’ in our large database of 688 landraces by 24 SSR makers. With only 12 of the 24 SSR markers assayed, all of the samples could be rejected as possible hybrids with ‘Yungay’ as a parent. This result demonstrates that the unintentional introduction of a transgene, not under farmers’ selection, from a widely grown transgenic variety over a long period of time is unlikely to happen at a detectable scale. Our finding reinforces the prominent role of farmers in the selection and maintenance of landraces which, unlike hybrids, have specific characteristics that farmers appreciate.

Harnessing oncolytic virus-mediated anti-tumor immunity.

Harnessing oncolytic virus-mediated anti-tumor immunity.

Gateway®-compatible plant transformation vectors.

Studies in functional genomics and crop improvement programs often rely on the introduction and expression of transgenes in plants. There are two essential components required for in planta transgene expression, a plasmid vector on which the transgene sequence is carried and a delivery system capable of transferring the vector to the target cells. Agrobacterium-mediated plant transformation and the binary plasmid vector system is the preferred method of transgene delivery. The cloning technologies used for DNA manipulation underpin many of these studies. Increased demand for efficient high-throughput transformation systems is driving forward improvements in gene cloning techniques. This chapter gives an overview of Gateway(®)-compatible binary vectors for use in Agrobacterium-mediated transformation systems. It describes a fast, efficient, and robust cloning protocol for the production of an over-expression binary vector using Gateway(®) recombinational cloning.

WNT3A promotes myogenesis of human embryonic stem cells and enhances in vivo engraftment

The ability of human embryonic stem cells (hESCs) to differentiate into skeletal muscle cells is an important criterion in using them as a cell source to ameliorate skeletal muscle impairments. However, differentiation of hESCs into skeletal muscle cells still remains a challenge, often requiring introduction of transgenes. Here, we describe the use of WNT3A protein to promote in vitro myogenic commitment of hESC-derived cells and their subsequent in vivo function. Our findings show that the presence of WNT3A in culture medium significantly promotes myogenic commitment of hESC-derived progenitors expressing a mesodermal marker, platelet-derived growth factor receptor-α (PDGFRA), as evident from the expression of myogenic markers, including DES, MYOG, MYH1, and MF20. In vivo transplantation of these committed cells into cardiotoxin-injured skeletal muscles of NOD/SCID mice reveals survival and engraftment of the donor cells. The cells contributed to the regeneration of damaged muscle fibers and the satellite cell compartment. In lieu of the limited cell source for treating skeletal muscle defects, the hESC-derived PDGFRA+ cells exhibit significant in vitro expansion while maintaining their myogenic potential. The results described in this study provide a proof-of-principle that myogenic progenitor cells with in vivo engraftment potential can be derived from hESCs without genetic manipulation.

Sources of CNS tumor heterogeneity

Central nervous system tumors are marked with high intra- and inter-tumoral heterogeneity. For instance, an integrative large scale gene expression study performed in 2010 revealed 4 subtypes glioblastoma multiforme (GBM) with differential responses to treatment and prognosis [1]. A more comprehensive GBM classification based on combination of epigenetics, copy number variation, gene expression and genetic mutation analysis has led to identification of as many as 6 GBM subgroups [2]. Two, not mutually exclusive, general models have been proposed to explain tumor heterogeneity [3]. The genetic mutation model proposes that different genetic mutations lead to different tumor formation, while the cell of origin model explains different tumors as arising from different cell types. There is experimental evidence supporting both models. For example, 3 distinctly different CNS tumor types can be induced by infection of postnatal mouse neural stem cells with virus containing V12HRAS and c-MYC depending on the combination and sequence in which oncogenes are introduced [4]. Similarly, RNA interference (RNAi) knock down of NF1 and p53 in GFAP+ or SynI+ cells induces mesenchymal GBM, whereas the same RNAi in Nestin+ cells induced neural GBM[5]. GMB gene expression analysis also indicates that different GBM subtypes have transcipt profiles similar to different cell types [1]. To fully explore the causes of tumor diversity, it is desirable2 to have an animal model in which both the cell of origin and genetic insult can be conveniently and independently manipulated. To achieve this, we have recently developed a central nervous system tumor model in the rat in which multiple oncogenes can be expressed in selected cell populations at different times in brain development [6]. In this model, we used the piggyBac transposon system [7] to stably integrate oncogenes into defined cell populations by in utero electroporation (IUE). Using this model, we evaluated the contribution of cell of origin and genetic mutation in tumor heterogeneity. To test whether the same oncogenic event in different, but closely related, cell population gives rise to same or different tumors, we directed HRasV12/AKT expression in disparate cell populations in the radial glia lineage with promoters that are ubiquitously active (CAG promoter), astrocyte selective GFAP (glial fibrillary acidic protein) promoter or oligodendrocytes selective MBP (myelin basic protein) promoter. We showed that HRasV12/AKT expression under CAG or GFAP promoter induced similar tumors, glioblastoma multiforme (WHO grade 4). However, HRasV12/AKT expression controlled by MBP promoter induced anaplastic oligoastrocytoma (WHO grade 3). We further showed that these induced induced anaplastic oligoastrocytoma differed from glioblastoma multiforme both in histology and molecular signature. These results indicate that oncogenic events occurring in different cell types in the same cellular lineage can lead to different tumor types. We next investigated whether tumor phenotype could be modified by expression of neurogenic bHLH family protein Neurogenin2 (Ngn2) or Neural differentiation 1 (NeuroD1). Members of the bHLH gene family are well known to have important roles is cell-type determination in normal development. Expression of either Ngn2 or NeuroD1 along with HRasV12/AKT resulted in atypical teratoid rhabdoid tumor like (ATRT like) tumor, a tumor type not previously observed after expression of HRasV12/AKT oncogenes alone. We further tested whether this phenotypic transformation from GBM to ATRT like tumor was due to transient expression of bHLH factors in radial glia or due to expression in tumor cells. Our data showed that transient expression of Ngn2 in radial glia, prior to transformation by HRasV12/AKT was able to induce ATRT like tumor formation. These results may indicate that the same oncogenic events occurring in similar cell types expressing different levels of individual bHLH transcription factors can lead to very different tumor types. The main advantage of piggyBac IUE method is that it allows for introduction of multiple transgenes controlled by independent promoters. The high co-expression rates allowed us to direct expression in different subpopulations in sequence. In the same system, we also demonstrated use of multi-color fluorescent protein expression to produce a clonal readout of tumor growth and invasion. There are several additional features of the piggyBac system that make it useful for other applications in investigating tumor biology. For example, the piggyBac- IUE approach could be applied to other species other than rodent extending CNS tumor biology to other species, such as ferret. Combination of piggyBac-IUE with existing transgenic mouse models could potentially broaden the utility of both approaches. Also multicolor clonal labeling of tumor cells could facilitate in vivo imaging of clonally related tumor cells. These functionalities should make this approach a useful platform for screening potential modifiers of tumor development and for studying further how genetic modifiers and cell or origin are related to tumor development and heterogeneity.

RNAi-mediated gene silencing in zebrafish triggered by convergent transcription.

RNAi based strategies to induce gene silencing are commonly employed in numerous model organisms but have not been extensively used in zebrafish. We found that introduction of transgenes containing convergent transcription units in zebrafish embryos induced stable transcriptional gene silencing (TGS) in cis and trans for reporter (mCherry) and endogenous (One-Eyed Pinhead (OEP) and miR-27a/b) genes. Convergent transcription enabled detection of both sense and antisense transcripts and silencing was suppressed upon Dicer knockdown, indicating processing of double stranded RNA. By ChIP analyses, increased silencing was accompanied by enrichment of the heterochromatin mark H3K9me3 in the two convergently arranged promoters and in the intervening reading frame. Our work demonstrates that convergent transcription can induce gene silencing in zebrafish providing another tool to create specific temporal and spatial control of gene expression.

In vivo introduction of transgenes into mouse sciatic nerve cells in situ using viral vectors.

The myelin sheath is essential for the rapid and efficient propagation of action potentials. However, our understanding of the basic molecular mechanisms that regulate myelination, demyelination and remyelination is limited. Schwann cells produce myelin in the peripheral nervous system and remain associated with the axons of peripheral neurons throughout axonal migration to the target. Owing to the intimate relationship between these cell types it is difficult to fully reproduce their function in vitro. For this reason, we developed an approach based on the injection of an engineered virus into the sciatic nerve of mice to locally transduce peripheral nerve cells. This approach can be used as an alternative to germline transgenesis to facilitate the investigation of peripheral nerve biology in vivo. The detailed protocol, described here, requires 3 weeks to complete. In comparison with genetic modification strategies, this protocol is a fast, reproducible and straightforward method for introducing exogenous factors into myelinating Schwann cells and myelinated axons in vivo to investigate specific molecular mechanisms.

A histone methyltransferase inhibits seed germination by increasing PIF1 mRNA expression in imbibed seeds

Phytochrome-interacting factor 1 (PIF1) inhibits light-dependent seed germination. The specific function of PIF1 in seed germination is partly due to its high level of expression in imbibed seeds, but the associated regulatory factors have not been identified. Here we show that mutation of the early flowering in short days (EFS) gene, encoding an H3K4 and H3K36 methyltransferase, decreases the level of H3K36me2 and H3K36me3 but not H3K4me3 at the PIF1 locus, reduces the targeting of RNA polymeraseII to the PIF1 locus, and reduces mRNA expression of PIF1 in imbibed seeds. Consistently, the efs mutant geminated even under the phyBoff condition, and had an expression profile of PIF1 target genes similar to that of the pif1 mutant. Introduction of an EFS transgene into the efs mutant restored the level of H3K36me2 and H3K36me3 at the PIF1 locus, the high-level expression of PIF1mRNA, the expression pattern of PIF1 target genes, and the light-dependent germination of these seeds. Introduction of a PIF1 transgene into the efs mutant also restored the expression pattern of PIF1 target genes and light-dependent germination in imbibed seeds, but did not restore the flowering phenotype. Taken together, our results indicate that EFS is necessary for high-level expression of PIF1mRNA in imbibed seeds.

Matrix Metalloproteinase-20 Over-Expression Is Detrimental to Enamel Development: A Mus musculus Model

BackgroundMatrix metalloproteinase-20 (Mmp20) ablated mice have enamel that is thin and soft with an abnormal rod pattern that abrades from the underlying dentin. We asked if introduction of transgenes expressing Mmp20 would revert this Mmp20 null phenotype back to normal. Unexpectedly, for transgenes expressing medium or high levels of Mmp20, we found opposite enamel phenotypes depending on the genetic background (Mmp20−/− or Mmp20+/+) in which the transgenes were expressed.Methodology/Principal Findings Amelx-promoter-Mmp20 transgenic founder mouse lines were assessed for transgene expression and those expressing low, medium or high levels of Mmp20 were selected for breeding into the Mmp20 null background. Regardless of expression level, each transgene brought the null enamel back to full thickness. However, the high and medium expressing Mmp20 transgenes in the Mmp20 null background had significantly harder more mineralized enamel than did the low transgene expresser. Strikingly, when the high and medium expressing Mmp20 transgenes were present in the wild-type background, the enamel was significantly less well mineralized than normal. Protein gel analysis of enamel matrix proteins from the high and medium expressing transgenes present in the wild-type background demonstrated that greater than normal amounts of cleavage products and smaller quantities of higher molecular weight proteins were present within their enamel matrices.Conclusions/Significance Mmp20 expression levels must be within a specific range for normal enamel development to occur. Creation of a normally thick enamel layer may occur over a wider range of Mmp20 expression levels, but acquisition of normal enamel hardness has a narrower range. Since over-expression of Mmp20 results in decreased enamel hardness, this suggests that a balance exists between cleaved and full-length enamel matrix proteins that are essential for formation of a properly hardened enamel layer. It also suggests that few feedback controls are present in the enamel matrix to prevent excessive MMP20 activity.

Prostate Epithelium-Specific Deletion of the Selenocysteine tRNA Gene Trsp Leads to Early Onset Intraepithelial Neoplasia

Although various lines of evidence suggest that oxidative stress plays a role in human prostate cancer initiation and progression, there is a paucity of direct evidence for its role in tumor initiation. To begin to address this issue, we developed a novel tumorigenesis model by reducing the expression of multiple selenoproteins (SPs) in mouse prostatic epithelium. This was accomplished via the prostate-specific deletion of Trsp, a gene that encodes a transfer RNA (Sec tRNA) required for the insertion of selenocysteine residues into SPs during their translation. By 6 weeks of age, Trsp-deficient mice exhibited widespread prostatic intraepithelial neoplasia lesions in all prostatic lobes, which then progressed to high-grade dysplasia and microinvasive carcinoma by 24 weeks. In contrast to other murine prostate cancer models, Trsp-deficient mice required neither the deletion of a tumor suppressor nor the transgenic introduction of an oncogene for prostatic intraepithelial neoplasia lesion development. In keeping with the antioxidant functions of several SPs, we found increases in lipid peroxidation markers in Trsp-deficient epithelial cells. This novel model of prostate neoplasia provides evidence for the existence of a selenoprotein or selenoproteins capable of acting as a tumor suppressor in the murine prostate.

Rapid target gene validation in complex cancer mouse models using re‐derived embryonic stem cells

Human cancers modeled in Genetically Engineered Mouse Models (GEMMs) can provide important mechanistic insights into the molecular basis of tumor development and enable testing of new intervention strategies. The inherent complexity of these models, with often multiple modified tumor suppressor genes and oncogenes, has hampered their use as preclinical models for validating cancer genes and drug targets. In our newly developed approach for the fast generation of tumor cohorts we have overcome this obstacle, as exemplified for three GEMMs; two lung cancer models and one mesothelioma model. Three elements are central for this system; (i) The efficient derivation of authentic Embryonic Stem Cells (ESCs) from established GEMMs, (ii) the routine introduction of transgenes of choice in these GEMM-ESCs by Flp recombinase-mediated integration and (iii) the direct use of the chimeric animals in tumor cohorts. By applying stringent quality controls, the GEMM-ESC approach proofs to be a reliable and effective method to speed up cancer gene assessment and target validation. As proof-of-principle, we demonstrate that MycL1 is a key driver gene in Small Cell Lung Cancer.

Impact of mating behaviour on the success of malaria control through a single inundative release of transgenic mosquitoes

Transgenic mosquitoes are a potential tool for the control or eradication of insect-vectored diseases. For malaria, one possible strategy relies on the introduction of malaria-refractory transgenes into wild Anopheles mosquito populations that would limit their capacity to transmit the disease. The success of such an approach obviously depends on a variety of factors. By developing a model that integrates both population genetics and epidemiology, we explore how mosquito mating preferences and the cost and efficacy of refractoriness affects the long-term prevalence of malaria in humans subsequent to a single generation inundative release of male transgenic mosquitoes. As may be intuitively expected, mating discrimination by wild-type individuals against transgenic ones generally reduces the probability that transgenes become stably established at a high frequency in mosquito populations. We also show that in circumstances where transgenic individuals exhibit some degree of discrimination against wild-type individuals, this can favour the spread of refractory alleles and lead to a significant reduction in malaria prevalence in the human population (if the efficacy of a dominant refractory mechanism exceeds at least 75%). The existence of such a non-intuitive outcome highlights the practical value of increasing the understanding of Anopheles mating preferences in the wild as a means to harness them in the implementation of population replacement approaches. Potential strategies by which previously described mating preferences of Anopheles gambiae populations could be exploited to manipulate the mate choice of transgenic release stocks are discussed.

Mutational Breeding and Genetic Engineering in the Development of High Grain Protein Content

Cereals are the most important crops in the world for both human consumption and animal feed. Improving their nutritional values, such as high protein content, will have significant implications, from establishing healthy lifestyles to helping remediate malnutrition problems worldwide. Besides providing a source of carbohydrate, grain is also a natural source of dietary fiber, vitamins, minerals, specific oils, and other disease-fighting phytocompounds. Even though cereal grains contain relatively little protein compared to legume seeds, they provide protein for the nutrition of humans and livestock that is about 3 times that of legumes. Most cereal seeds lack a few essential amino acids; therefore, they have imbalanced amino acid profiles. Lysine (Lys), threonine (Thr), methionine (Met), and tryptophan (Trp) are among the most critical and are a limiting factor in many grain crops for human nutrition. Tremendous research has been put into the efforts to improve these essential amino acids. Development of high protein content can be outlined in four different approaches through manipulating seed protein bodies, modulating certain biosynthetic pathways to overproduce essential and limiting amino acids, increasing nitrogen relocation to the grain through the introduction of transgenes, and exploiting new genetic variance. Various technologies have been employed to improve protein content including conventional and mutational breeding, genetic engineering, marker-assisted selection, and genomic analysis. Each approach involves a combination of these technologies. Advancements in nutrigenomics and nutrigenetics continue to improve public knowledge at a rapid pace on the importance of specific aspects of food nutrition for optimum fitness and health. An understanding of the molecular basis for human health and genetic predisposition to certain diseases through human genomes enables individuals to personalize their nutritional requirements. It is critically important, therefore, to improve grain protein quality. Highly nutritious grain can be tailored to functional foods to meet the needs for both specific individuals and human populations as a whole.

122 : Regulation of thymocyte development by protein phosphatase 4

TCR repertoire is regulated sequentially by three major factors: VDJ recombination, thymic selection, and peripheral homeostasis. Using mice with Lck-cre-driven conditional deletion of PP4 locus, we show that protein phosphatase 4 (PP4), the product of PP4 gene, is essential for the development of mature T cells, such that thymocyte development is completely blocked at the CD4−CD8− stage in the absence of PP4 protein. Further studies of PP4’s functions during late thymocyte development and in mature T cells are, however, prevented by this severe developmental block. To circumvent this limitation, we introduced CD4-cre transgene into mice carrying the conditional PP4 allele (CD4cre-PP4f/f mice). FACS analyses and mixed bone marrow chimera experiments showed that thymocyte development was retarded at multiple stages in CD4cre-PP4f/f mice. The introduction of αβ TCR transgene could partially rescue thymocyte maturation, suggesting that PP4 may be important for the generation of TCR-expressing cells. Recently PP4 was shown to regulate γH2AX during DNA repair, which shared many components with VDJ recombination. Western and immunofluorescence analyses of PP4-deficient thymocytes, however, did not reveal any defect in H2AX phosphorylation. To further study the roles of PP4 in TCR generation, we resorted to an inducible VDJ recombination system that utilized vAbl-transformed cell lines. Results from this inducible system showed that the inhibition of PP4 did not cause defects in RAG expression or DNA repair, implying that VDJ recombination per se was not tightly regulated by PP4. Instead, PP4 inhibitor significantly enhanced the induction of apoptosis in cells with on-going VDJ recombination; interestingly, ATM antagonized the effects of PP4 in this context. Our studies thus reveal a novel role of PP4 in thymocytes maturation and TCR repertoire formation, in which the survival window of thymocytes with active VDJ recombination may be critically regulated by PP4.

Transcription activator like effector (TALE)-directed piggyBac transposition in human cells

Insertional therapies have shown great potential for combating genetic disease and safer methods would undoubtedly broaden the variety of possible illness that can be treated. A major challenge that remains is reducing the risk of insertional mutagenesis due to random insertion by both viral and non-viral vectors. Targetable nucleases are capable of inducing double-stranded breaks to enhance homologous recombination for the introduction of transgenes at specific sequences. However, off-target DNA cleavages at unknown sites can lead to mutations that are difficult to detect. Alternatively, the piggyBac transposase is able perform all of the steps required for integration; therefore, cells confirmed to contain a single copy of a targeted transposon, for which its location is known, are likely to be devoid of aberrant genomic modifications. We aimed to retarget transposon insertions by comparing a series of novel hyperactive piggyBac constructs tethered to a custom transcription activator like effector DNA-binding domain designed to bind the first intron of the human CCR5 gene. Multiple targeting strategies were evaluated using combinations of both plasmid-DNA and transposase-protein relocalization to the target sequence. We demonstrated user-defined directed transposition to the CCR5 genomic safe harbor and isolated single-copy clones harboring targeted integrations.

Complete TCR-α Gene Locus Control Region Activity in T Cells Derived In Vitro from Embryonic Stem Cells

Locus control regions (LCRs) are cis-acting gene regulatory elements with the unique, integration site-independent ability to transfer the characteristics of their locus-of-origin's gene expression pattern to a linked transgene in mice. LCR activities have been discovered in numerous T cell lineage-expressed gene loci. These elements can be adapted to the design of stem cell gene therapy vectors that direct robust therapeutic gene expression to the T cell progeny of engineered stem cells. Currently, transgenic mice provide the only experimental approach that wholly supports all the critical aspects of LCR activity. In this study, we report the manifestation of all key features of mouse TCR-α gene LCR function in T cells derived in vitro from mouse embryonic stem cells. High-level, copy number-related TCR-α LCR-linked reporter gene expression levels are cell type restricted in this system, and upregulated during the expected stage transition of T cell development. We also report that de novo introduction of TCR-α LCR-linked transgenes into existing T cell lines yields incomplete LCR activity. These data indicate that establishing full TCR-α LCR activity requires critical molecular events occurring prior to final T lineage determination. This study also validates a novel, tractable, and more rapid approach for the study of LCR activity in T cells, and its translation to therapeutic genetic engineering.

Hereditary cataract of the Nakano mouse: Involvement of a hypomorphic mutation in the coproporphyrinogen oxidase gene

The Nakano cataract (NCT) is a recessive disorder in the mouse linked to the nct locus on chromosome 16. In this study, we positionally cloned the critical gene in the nct locus. Herein, we report that cataracts in the BALB/c-nct/nct mouse are caused by a hypomorphic mutation in the coproporphyrin oxidase gene (Cpox), encoding the enzyme responsible for catalyzing oxidative decarboxylation of the heme precursor, coproporphyrinogen III, in the heme biosynthetic pathway. BALB/c-nct/nct mice are homozygous for a G to T nucleotide substitution in the Cpox gene, which results in a p.R380L amino acid substitution in the CPOX protein. The CPOX isoform with the p.R380L substitution retained only 15% of the activity of the wild type isoform. BALB/c-nct/nct mice had excessive accumulation of coproporphyrin III in the lens. The NCT phenotype was normalized by the introduction of a wild type Cpox transgene. The mechanisms by which impairment of CPOX leads to lens opacity in the NCT are elusive. However, our data illuminate a hitherto unanticipated involvement of the heme biosynthesis pathway in lens physiology.

Cynomolgus monkey induced pluripotent stem cells established by using exogenous genes derived from the same monkey species

Induced pluripotent stem (iPS) cells established by introduction of the transgenes POU5F1 (also known as Oct3/4), SOX2, KLF4 and c-MYC have competence similar to embryonic stem (ES) cells. iPS cells generated from cynomolgus monkey somatic cells by using genes taken from the same species would be a particularly important resource, since various biomedical investigations, including studies on the safety and efficacy of drugs, medical technology development, and research resource development, have been performed using cynomolgus monkeys. In addition, the use of xenogeneic genes would cause complicating matters such as immune responses when they are expressed. In this study, therefore, we established iPS cells by infecting cells from the fetal liver and newborn skin with amphotropic retroviral vectors containing cDNAs for the cynomolgus monkey genes of POU5F1, SOX2, KLF4 and c-MYC. Flat colonies consisting of cells with large nuclei, similar to those in other primate ES cell lines, appeared and were stably maintained. These cell lines had normal chromosome numbers, expressed pluripotency markers and formed teratomas. We thus generated cynomolgus monkey iPS cell lines without the introduction of ecotropic retroviral receptors or other additional transgenes by using the four allogeneic transgenes. This may enable detailed analysis of the mechanisms underlying the reprogramming. In conclusion, we showed that iPS cells could be derived from cynomolgus monkey somatic cells. To the best of our knowledge, this is the first report on iPS cell lines established from cynomolgus monkey somatic cells by using genes from the same species.

Decreased proteoglycan degradation in IL-1β-treated cartilage co-cultured with TIMP-3-transduced cells

Purpose: In the degenerative environment of osteoarthritis (OA), the enzymes responsible for the degradation of aggrecan, a major proteoglycan of cartilage are up-regulated. These aggrecanases degrade the glycosaminoglycans (GAGs) within the proteoglycan protein. Tissue Inhibitor of Metalloproteinases (TIMPs) normally maintain this enzyme activity within normal limits, but increased production of aggrecanases has been observed in cartilage affected by OA. In the current experiment, we up-regulated full-length TIMP-3 protein signaling in situ, through introduction of a TIMP-3 transgene in a monolayer-cell/cartilage-tissue co-culture model. We hypothesized that in situ up-regulation of cell-produced, full-length TIMP-3 protein would decrease proteoglycan degradation. Methods: We co-cultured cartilage biopsies with TIMP-3-stably-transduced HEK cells and recombinant adeno-associated viral vector (rAAV-TIMP-3)-transduced equine fetal fibroblasts. TIMP-3 cells were plated in 12-well co-culture plates at 1.0E+05 cells per well in serum-free media. Equine cartilage biopsy tissue was added to the plates with or without IL-1β (10ng/ml) and with or without rhTIMP-3 protein (100ng/ml). Media and cartilage samples were collected after being exposure to a protease inhibitor and were stored at -80°C until analyzed. Glycosaminoglycan content was analyzed with a standard DMMB assay. Aggrecanase activity was analyzed with a Sensitive Aggrecanase ELISA assay. Results: In four days of serum-free culture, the amount of GAG remaining in cartilage biopsies was increased by culturing the cartilage with rhTIMP-3 protein. Co-culturing the cartilage biopsies with HEK-TIMP-3 cells and rAAV-TIMP-3-transduced fetal fibroblasts further increased the percentage of GAG retained in the cartilage. Aggrecanase activity, as measured by the concentration of ARGSVIL aggrecan fragments in co-culture conditioned media was decreased by the presence of TIMP-3 cells. Conclusions: Preservation of GAG in cartilage biopsy tissue was observed when the tissue was co-cultured with 1) a stably-transduced TIMP-3-HEK cell line, 2) with rAAV-TIMP-3-transduced fetal fibroblasts and 3) with rhTIMP-3 protein. TIMP-3-transduced cells produced protective effects greater than rhTIMP-3 protein alone. These observations present the possibilities of an in vivo therapeutic treatment with a rAAV-TIMP-3 vector alone or with transplantation of TIMP-3-transduced cells.Figure 2Glycosaminoglycan changes in articular cartilage exposed to TIMP-3. Co-culture with TIMP-3-transduced cells significantly increased the amount of GAG contained in IL-1β-cultured cartilage. Error bars represent SE.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 3Aggrecan changes in articular cartilage exposed to TIMP-3. Co-culture with TIMP-3-transduced cells significantly decreased the amount of the degraded -aggrecan fragment, ARGSVIL released into the media of IL-1β-cultured cartilage. Error bars represent SE.View Large Image Figure ViewerDownload Hi-res image Download (PPT)