RESUMEN

The Coffea spp. plant is a significant crop in Latin America, Africa, and Asia, and recent advances in genomics and transcriptomics have opened possibilities for studying candidate genes and introducing new desirable traits through genetic engineering. While stable transformation of coffee plants has been reported using various techniques, it is a time-consuming and laborious process. To overcome this, transient transformation methods have been developed, which avoid the limitations of stable transformation. This chapter describes an ex vitro protocol for transient expression using A. tumefaciens-mediated infiltration of coffee leaves, which could be used to produce coffee plants expressing desirable traits against biotic and abiotic stresses, genes controlling biochemical and physiological traits, as well as for gene editing through CRISPR/Cas9.

Asunto(s)

Agrobacterium tumefaciens , Coffea , Edición Génica , Hojas de la Planta , Plantas Modificadas Genéticamente , Transgenes , Coffea/genética , Hojas de la Planta/genética , Hojas de la Planta/metabolismo , Plantas Modificadas Genéticamente/genética , Agrobacterium tumefaciens/genética , Edición Génica/métodos , Transformación Genética , Sistemas CRISPR-Cas , Regulación de la Expresión Génica de las Plantas

RESUMEN

Recombinant adeno-associated viral vectors (rAAV) are the safest and most effective gene delivery platform to drive the treatment of many inherited eye disorders in well-characterized animal models. The use in rAAV of ubiquitous promoters derived from viral sequences such as CMV/CBA (chicken ß-actin promoter with cytomegalovirus enhancer) can lead to unwanted side effects such as pro-inflammatory immune responses and retinal cytotoxicity, thus reducing therapy efficacy. Thus, an advance in gene therapy is the availability of small promoters, that potentiate and direct gene expression to the cell type of interest, with higher safety and efficacy. In this study, we used six human mini-promoters packaged in rAAV2 quadruple mutant (Y-F) to test for transduction of the rat retina after intravitreal injection. After four weeks, immunohistochemical analysis detected GFP-labeled cells in the ganglion cell layer (GCL) for all constructs tested. Among them, Ple25sh1, Ple25sh2 and Ple53 promoted a widespread reporter-transgene expression in the GCL, with an increased number of GFP-expressing retinal ganglion cells when compared with the CMV/CBA vector. Moreover, Ple53 provided the strongest levels of GFP fluorescence in both cell soma and axons of retinal ganglion cells (RGCs) without any detectable adverse effects in retina function. Remarkably, a nearly 50-fold reduction in the number of intravitreally injected vector particles containing Ple53 promoter, still attained levels of transgene expression similar to CMV/CBA. Thus, the tested MiniPs show great potential for protocols of retinal gene therapy in therapeutic applications for retinal degenerations, especially those involving RGC-related disorders such as glaucoma.

Asunto(s)

Infecciones por Citomegalovirus , Células Ganglionares de la Retina , Ratas , Humanos , Animales , Células Ganglionares de la Retina/metabolismo , Vectores Genéticos , Retina/metabolismo , Transgenes , Inyecciones Intravítreas , Infecciones por Citomegalovirus/genética , Infecciones por Citomegalovirus/metabolismo , Dependovirus/genética , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Transducción Genética

RESUMEN

The presence and levels of transgenic maize in Mexico and the effect this could have on local landraces or closely related species such as teosinte has been the subject of several previous reports, some showing contrasting results. Cultural, social and political factors all affect maize cultivation in Mexico and although since 1998 there has been a moratorium on the commercial cultivation of transgenic maize, Mexico imports maize, mainly from the USA where transgenic cultivars are widely grown. Additionally extensive migration between rural areas in Mexico and the USA and customs of seed exchange between farmers may also play an unintentional role in the establishment of transgenic seed. A comprehensive study of all Mexican maize landraces throughout the country is not feasible, however this report presents data based on analysis of 3204 maize accessions obtained from the central region of Mexico (where permits have never been authorized for cultivation of transgenic maize) and the northern region (where for a short period authorization for experimental plots was granted). The results of the study confirm that transgenes are present in all the geographical areas sampled and were more common in germplasm obtained in the northern region. However, there was no evidence that regions where field trials had been authorized showed higher levels of transgene presence or that the morphology of seed lots harboring transgenic material was significantly modified in favor of expected transgenic phenotypes.

Asunto(s)

Zea mays , Animales , Plantas Modificadas Genéticamente/genética , Zea mays/genética , México , Transgenes , Animales Modificados Genéticamente

RESUMEN

Regulated systems for transgene expression are useful tools in basic research and a promising platform in biomedicine due to their regulated transgene expression by an inducer. The emergence of optogenetics expression systems enabled the construction of light-switchable systems, enhancing the spatial and temporal resolution of a transgene. The LightOn system is an optogenetic tool that regulates the expression of a gene of interest using blue light as an inducer. This system is based on a photosensitive protein (GAVPO), which dimerizes and binds to the UASG sequence in response to blue light, triggering the expression of a downstream transgene. Previously, we adapted the LightOn system to a dual lentiviral vector system for neurons. Here, we continue the optimization and assemble all components of the LightOn system into a single lentiviral plasmid, the OPTO-BLUE system. For functional validation, we used enhanced green fluorescent protein (EGFP) as an expression reporter (OPTO-BLUE-EGFP) and evaluated the efficiency of EGFP expression by transfection and transduction in HEK293-T cells exposed to continuous blue-light illumination. Altogether, these results prove that the optimized OPTO-BLUE system allows the light-controlled expression of a reporter protein according to a specific time and light intensity. Likewise, this system should provide an important molecular tool to modulate gene expression of any protein by blue light.

Asunto(s)

Vectores Genéticos , Optogenética , Humanos , Optogenética/métodos , Células HEK293 , Transfección , Transgenes , Expresión Génica , Vectores Genéticos/genética , Lentivirus/genética

RESUMEN

Agrobacterium rhizogenes-mediated transformation has long been explored as a versatile and reliable method for gene function validation in many plant species, including soybean (Glycine max). Likewise, detached-leaf assays have been widely used for rapid and mass screening of soybean genotypes for disease resistance. The present study combines these two methods to establish an efficient and practical system to generate transgenic soybean hairy roots from detached leaves and their subsequent culture under ex vitro conditions. We demonstrated that hairy roots derived from leaves of two (tropical and temperate) soybean cultivars could be successfully infected by economically important species of root-knot nematodes (Meloidogyne incognita and M. javanica). The established detached-leaf method was further explored for functional validation of two candidate genes encoding for cell wall modifying proteins (CWMPs) to promote resistance against M. incognita through distinct biotechnological strategies: the overexpression of a wild Arachis α-expansin transgene (AdEXPA24) and the dsRNA-mediated silencing of an endogenous soybean polygalacturonase gene (GmPG). AdEXPA24 overexpression in hairy roots of RKN-susceptible soybean cultivar significantly reduced nematode infection by approximately 47%, whereas GmPG downregulation caused an average decrease of 37%. This novel system of hairy root induction from detached leaves showed to be an efficient, practical, fast, and low-cost method suitable for high throughput in root analysis of candidate genes in soybean.

Asunto(s)

Glycine max , Nematodos , Animales , Glycine max/genética , Glycine max/metabolismo , Nematodos/genética , Transgenes , Hojas de la Planta/genética , Hojas de la Planta/metabolismo , Genotipo

RESUMEN

Dengue viruses (DENVs) are mosquito-borne flaviviruses causing millions of human infections each year and pose a challenge for public health systems worldwide. Aedes aegypti is the principal vector species transmitting DENVs to humans. Controlling Ae. aegypti is difficult due to the abundance of breeding sites and increasing insecticide resistance in the vector populations. Developing new vector control strategies is critical for decreasing the disease burden. One potential approach is genetically replacing Ae. aegypti populations with vector populations highly resistant to DENV transmission. Here, we focus on an alternative strategy for generating dengue 2 virus (DENV-2) resistance in genetically-modified Ae. aegypti in which the mosquitoes express an inactive form of Michelob_x (Mx), an antagonist of the Inhibitor of Apoptosis (IAP), to induce apoptosis in those cells in which actively replicating DENV-2 is present. The inactive form of Mx was flanked by the RRRRSAG cleavage motif, which was recognized by the NS2B/NS3 protease of the infecting DENV-2 thereby releasing and activating Mx which then induced apoptosis. Our transgenic strain exhibited a significantly higher mortality rate than the non-transgenic control when infected with DENV-2. We also transfected a DNA construct containing inactive Mx fused to eGFP into C6/36 mosquito cells and indirectly observed Mx activation on days 3 and 6 post-DENV-2 infections. There were clear signs that the viral NS2B/NS3 protease cleaved the transgene, thereby releasing Mx protein into the cytoplasm, as was confirmed by the detection of eGFP expression in infected cells. The present study represents proof of the concept that virus infection can be used to induce apoptosis in infected mosquito cells.

Asunto(s)

Aedes , Virus del Dengue , Dengue , Animales , Humanos , Virus del Dengue/genética , Muerte Celular , Transgenes , Péptido Hidrolasas/genética

RESUMEN

We aimed to assess the potential of baculoviral vectors (BV) for brain cancer gene therapy. We compared them with adenoviral vectors (AdV), which are used in neuro-oncology, but for which there is pre-existing immunity. We constructed BVs and AdVs encoding fluorescent reporter proteins and evaluated their transduction efficiency in glioma cells and astrocytes. Naïve and glioma-bearing mice were intracranially injected with BVs to assess transduction and neuropathology. Transgene expression was also assessed in the brain of BV-preimmunized mice. While the expression of BVs was weaker than AdVs in murine and human glioma cell lines, BV-mediated transgene expression in patient-derived glioma cells was similar to AdV-mediated transduction and showed strong correlation with clathrin expression, a protein that interacts with the baculovirus glycoprotein GP64, mediating BV endocytosis. BVs efficiently transduced normal and neoplastic astrocytes in vivo, without apparent neurotoxicity. BV-mediated transgene expression was stable for at least 21 days in the brain of naïve mice, but it was significantly reduced after 7 days in mice systemically preimmunized with BVs. Our findings indicate that BVs efficiently transduce glioma cells and astrocytes without apparent neurotoxicity. Since humans do not present pre-existing immunity against BVs, these vectors may constitute a valuable tool for the delivery of therapeutic genes into the brain.

Asunto(s)

Baculoviridae , Neoplasias Encefálicas , Terapia Genética , Vectores Genéticos , Glioma , Baculoviridae/genética , Baculoviridae/inmunología , Neoplasias Encefálicas/terapia , Glioma/terapia , Animales , Ratones , Línea Celular Tumoral , Humanos , Ratas , Ratones Endogámicos C57BL , Masculino , Transducción Genética , Astrocitos/virología , Transgenes/genética

RESUMEN

Pericytes and glial cells are known to collaborate in dental pulp tissue repair. Cell-based therapies that stimulate these stromal components may be of therapeutic relevance for partially vital dental pulp conditions. This study aimed to examine the early effect of photobiomodulation (PBM) in pericytes from experimentally injured pulp tissue. To accomplish this, we used the Nestin-GFP/NG2-DsRed mice, which could allow the identification of distinct pericyte phenotypes. We discovered the presence of two pericytes subsets within the dental pulp, the Nestin + NG2+ (type-2) and Nestin- NG2+ (type-1). Upon injury, PBM treatment led to a significant increase in Nestin+ cells and pericytes. This boost was mainly conferred by the more committed pericyte subset (NestinNG2+ ). PBM also stimulated terminal blood vessels sprouting adjacent to the injury site while maintaining signs of pulp vitality. In vitro, PBM induced VEGF upregulation, improved dental pulp cells proliferation and migration, and favored their mineralization potential. Herein, different subsets of perivascular cells were unveiled in the pulp tissue. PBM enhanced not only NG2+ cells but nestin-expressing progenitors in the injured dental pulp.

Asunto(s)

Pulpa Dental/citología , Neuroglía , Pericitos , Animales , Ratones , Nestina/genética , Transgenes

RESUMEN

The Rad, Rem, Rem2, and Gem/Kir (RGK) sub-family of small GTP-binding proteins are crucial in regulating high voltage-activated (HVA) calcium channels. RGK proteins inhibit calcium current by either promoting endocytosis or reducing channel activity. They all can associate directly with Ca2+ channel ß subunit (CaVß), and the binding between CaVα1/CaVß appears essential for the endocytic promotion of CaV1.X, CaV2.1, and CaV2.2 channels. In this study, we investigated the inhibition of CaV2.3 channels by RGK proteins in the absence of CaVß. To this end, Xenopus laevis oocytes expressing CaV2.3 channels devoid of auxiliary subunit were injected with purified Gem and Rem and found that only Gem had an effect. Ca currents and charge movements were reduced by injection of Gem, pointing to a reduction in the number of channels in the plasma membrane. Since this reduction was ablated by co-expression of the dominant-negative mutant of dynamin K44A, enhanced endocytosis appears to mediate this reduction in the number of channels. Thus, Gem inhibition of CaV2.3 channels would be the only example of a CaVß independent promotion of dynamin-dependent endocytosis.

Asunto(s)

Potenciales de Acción/fisiología , Canales de Calcio Tipo R/genética , Proteínas de Transporte de Catión/genética , Dinaminas/genética , Proteínas de Unión al GTP Monoméricas/genética , Sustitución de Aminoácidos , Animales , Canales de Calcio Tipo R/metabolismo , Proteínas de Transporte de Catión/metabolismo , Dinaminas/metabolismo , Endocitosis/genética , Femenino , Expresión Génica , Humanos , Proteínas de Unión al GTP Monoméricas/metabolismo , Mutación , Oocitos/citología , Oocitos/metabolismo , Técnicas de Placa-Clamp , Plásmidos/química , Plásmidos/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transfección , Transgenes , Xenopus laevis

RESUMEN

Chronic liver injury leads to advanced fibrosis, cirrhosis, and hepatocellular carcinoma. Genetical cell treatment related to the use of adenovirus (Ads) has proven to be beneficial and efficient in the recovery of hepatic diseases. Nevertheless, they are highly immunogenic and trigger an immune response where interferons type 1 (IFN-I) play a very important role. Three shRNAs against the Interferon-1 receptor (IFNAR1) were designed and cloned in pENTR/U6 plasmid and amplified in DH5α cells. Huh7 cells were transfected with these plasmids in the presence or absence of 1 × 109 viral particles/ml of adenovirus containing the green fluorescent protein gene used as a reporter. Transfection with the shRNA plasmids partially inhibited the IFNAR1 expression. This inhibition substantially decreased antiviral response, demonstrated by the decrease of IFNAR1, IFN-α, and TNF-α gene expression, and the decrease at protein levels of IFNAR1, Protein kinase RNA-activated (PKR), and phosphorylated STAT1, allowing higher adenoviral transduction and transgene expression. Interestingly it was seen shRNA inhibited macrophage activation. These results suggest that the inhibition of the IFN-I pathway could be a strategy to minimize the immune response against Adenoviral vectors allowing higher Adenovirus transduction extending the transgene expression.

Asunto(s)

Adenoviridae , Receptor de Interferón alfa y beta , Adenoviridae/genética , Adenoviridae/metabolismo , Expresión Génica , Hepatocitos/metabolismo , ARN Interferente Pequeño/genética , Receptor de Interferón alfa y beta/genética , Receptor de Interferón alfa y beta/metabolismo , Transgenes

RESUMEN

Preserving morphological features that are important for cell function and structure is a critical parameter for in vitro experiments with rat cardiomyocytes. Lentiviral vectors are commonly used as gene transfer tool because of its high flexibility, efficiency to deliver expression cassettes and versatility of transducing quiescent cells. The tropism of the recombinant viral particle can be determined depending on the virus envelope, which shows a specific binding to cell surface receptors on the target cell. The combination of promoter arrangement and viral envelope must be optimized to achieve a greater transduction efficiency and a higher transgene expression. In this study we explored the optimization of promoters and heterologous envelopes to transduce primary culture of neonatal rat ventricular myocytes. Our results suggest a robust expression driven by the cytomegalovirus promoter, and high efficiency transduction mediated by VSV-G envelope with no apparent compromising ultrastructural features of genetically modified cells.

Asunto(s)

Lentivirus/genética , Miocitos Cardíacos/citología , Transducción Genética/métodos , Animales , Animales Recién Nacidos , Células Cultivadas , Citomegalovirus/genética , Expresión Génica , Vectores Genéticos , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Glicoproteínas de Membrana/genética , Miocitos Cardíacos/metabolismo , Regiones Promotoras Genéticas , Ratas , Sarcómeros/ultraestructura , Transgenes , Proteínas del Envoltorio Viral/genética , Pseudotipado Viral

RESUMEN

The availability of routine methods for the genetic engineering of the chloroplast genome of Chlamydomonas reinhardtii is allowing researchers to explore the use of this microalga as a phototrophic cell platform for synthesis of high value recombinant proteins and metabolites. However, the established method for delivering transforming DNA into the algal chloroplast involves microparticle bombardment using an expensive "gene gun". Furthermore, selection of transformant lines most commonly involves the use of a bacterial antibiotic resistance gene. In this chapter, we describe a simple and cheap delivery method in which cell-DNA suspensions are agitated with glass beads: a method that is more commonly used for nuclear transformation of Chlamydomonas. We also describe the use of plasmid expression vectors that target transgenes to a neutral site within the chloroplast genome between psbH and trnE2, and employ psbH as the selectable marker-thereby avoiding issues of unwanted antibiotic resistance genes in the resulting transgenic lines. Finally, we highlight a feature in our latest vectors in which the presence of a novel tRNA gene on the plasmid results in recognition within the chloroplast of UGA stop codons in transgenes as tryptophan codons. This feature simplifies the cloning of transgenes that are normally toxic to E. coli, serves as a biocontainment strategy restricting the functional escape of transgenes from the algal chloroplast to environmental microorganisms, and offers a simple system of temperature-regulated translation of transgenes.

Asunto(s)

Chlamydomonas reinhardtii/genética , Cloroplastos/genética , Ingeniería Genética/métodos , Plantas Modificadas Genéticamente/genética , Transformación Genética , Chlamydomonas reinhardtii/crecimiento & desarrollo , Vectores Genéticos , Genoma del Cloroplasto , Plantas Modificadas Genéticamente/crecimiento & desarrollo , Transgenes

RESUMEN

Obtaining data on transgene copy number is an integral step in the generation of transgenic plants. Techniques such as Southern blot, segregation analysis, and quantitative PCR (qPCR) have routinely been used for this task, in a range of species. More recently, use of Digital PCR (dPCR) has become prevalent, with a measurement accuracy higher than qPCR reported. Here, the relative merits of qPCR and dPCR for transgene copy number estimation in white clover were investigated. Furthermore, given that single copy reference genes are desirable for estimating gene copy number by relative quantification, and that no single-copy genes have been reported in this species, a search and evaluation of suitable reference genes in white clover was undertaken. Results demonstrated a higher accuracy of dPCR relative to qPCR for copy number estimation in white clover. Two genes, Pyruvate dehydrogenase (PDH), and an ATP-dependent protease, identified as single-copy genes, were used as references for copy number estimation by relative quantification. Identification of single-copy genes in white clover will enable the application of relative quantification for copy number estimation of other genes or transgenes in the species. The results generated here validate the use of dPCR as a reliable strategy for transgene copy number estimation in white clover, and provide resources for future copy number studies in this species.

Asunto(s)

Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Transgenes , Trifolium/genética , Variaciones en el Número de Copia de ADN , Dosificación de Gen , Hojas de la Planta/genética , Plantas Modificadas Genéticamente/genética

RESUMEN

As vantagens dos animais transgênicos têm sido demonstradas em diferentes aplicações, entretanto muitas metodologias usadas para gerar animais geneticamente modificados (GM) apresentam baixas taxas de eficiência. O objetivo deste estudo foi avaliar a entrega dos vetores lentivirais (VLs) em zigotos durante a fertilização in vitro (FIV), para gerar embriões GM, com o gene da proteína verde fluorescente (GFP) ou do fator IX de coagulação humana (FIX). Vetores lentivirais com os genes GFP (pLGW-GFP-LV) ou FIX (pLWE2-FIX-LV) foram utilizados na FIV ou na cultura de embriões in vitro (CIV). A coincubação de pLWE2-FIX-LV com espermatozoides e complexos oócitos-células do cumulus (COCs) durante a FIV diminuiu (P<0,05) as taxas de clivagem e de blastocistos, enquanto com pLGW-GFP-LV diminuiu (P<0,05) a taxa de blastocisto quando se comparou ao controle sem VL. A coincubação de pLWE2-FIX-LV e pLGW-GFP-LV com presumíveis zigotos durante a CIV não afetou (P>0,05) o desenvolvimento embrionário. A expressão da proteína GFP não foi detectada em embriões após a coincubação de FIV ou CIV, embora as células do cumulus expressassem a proteína até o dia oito de cultivo in vitro. Reações em cadeia da polimerase (PCR) não detectaram os genes GFP ou FIX em embriões, mas ambos foram detectados em células do cumulus. Assim, a coincubação de VL com espermatozoide bovino e COCs não é eficaz para produzir embriões geneticamente modificados por meio de FIV.(AU)

Asunto(s)

Animales , Bovinos , Cigoto , Animales Modificados Genéticamente/genética , Transgenes , Embrión de Mamíferos , Vectores Genéticos/análisis , Fertilización In Vitro/veterinaria , Técnicas de Transferencia de Gen/veterinaria

RESUMEN

After 25 years of genetically modified cotton cultivation in Mexico, gene flow between transgenic individuals and their wild relatives represents an opportunity for analysing the impacts of the presence of novel genes in ecological and evolutionary processes in natural conditions. We show comprehensive empirical evidence on the physiological, metabolic, and ecological effects of transgene introgression in wild cotton, Gossypium hirsutum. We report that the expression of both the cry and cp4-epsps genes in wild cotton under natural conditions altered extrafloral nectar inducibility and thus, its association with different ant species: the dominance of the defensive species Camponotus planatus in Bt plants, the presence of cp4-epsps without defence role of Monomorium ebeninum ants, and of the invasive species Paratrechina longicornis in wild plants without transgenes. Moreover, we found an increase in herbivore damage to cp4-epsps plants. Our results reveal the influence of transgene expression on native ecological interactions. These findings can be useful in the design of risk assessment methodologies for genetically modified organisms and the in situ conservation of G. hirsutum metapopulations.

Asunto(s)

Ecología , Evolución Molecular , Genes de Plantas , Gossypium/genética , Plantas Modificadas Genéticamente/genética , Transgenes , Animales , Hormigas/fisiología , Regulación de la Expresión Génica de las Plantas , Flujo Génico , Gossypium/fisiología , Herbivoria , México , Néctar de las Plantas

RESUMEN

In the context of comparative oncology, melanoma cells derived from companion animal tumors are good models for optimizing and predicting their in vivo response to therapeutic strategies. Here, we report that human, canine, and feline melanoma cells driven to death by bleomycin, interferon-ß gene, or herpes simplex virus thymidine kinase/ganciclovir suicide gene (SG) treatment significantly increased their internal granularity. This fact correlated with the release of a heterogeneous collection of nano- and micro-sized granules as revealed by transmission electron microscopy. While killing lipofected cells, the expressed transgenes and their derived products were incorporated into these granules that were isolated by differential centrifugation. These particulate factors (PFs) were able to transfer, in a dose- and time-dependent manner, appreciable levels of therapeutic genes, related proteins, and drugs. Thus, when recipient cells of SG-carrying PFs were exposed to ganciclovir, this prodrug was efficiently activated, eliminating them. These PFs kept the functionality of their cargo, even after being subjected to adverse conditions, such as the presence of DNase, freezing, or heating. Since our in vitro system did not include any of the immune mechanisms that could provide additional antitumor activity, the chemo-gene treatments amplified by these delivery bags of therapeutic agents offer a great clinical potential.

Asunto(s)

Interferón beta , Melanoma , Animales , Efecto Espectador , Gatos , Perros , Ganciclovir/farmacología , Humanos , Interferón beta/genética , Melanoma/genética , Melanoma/terapia , Simplexvirus/genética , Timidina Quinasa/genética , Transgenes

RESUMEN

The genotyping of genetically-modified cells is a crucial step in studies of transgenics and genomic editing with systems such as CRISPR/Cas. The detection of genome editing events can be directly related to the genotyping methodology used, which is influenced by its costs, since many experiments require the analysis of a large number of samples. The aim of this study was to compare the performance of direct lysis methods of genomic DNA (gDNA) extraction for the detection of knockins and knockouts in primary goat cells. Initially, three gDNA extraction protocols (protocol A, heat denaturation/freeze-thaw in water; protocol B, heat denaturation/proteinase K; and protocol C, CellsDirect Kit) were tested using different quantities (1,000, 5,000 and 10,000 cells) and types of goat primary cells (fibroblasts and goat mammary epithelial cells-GMECs) for subsequent validation by PCR amplification of small (GAPDH) and large amplicons (hLF transgene). All protocols were successful in the detection of the small amplicon; however, in GMECs, only protocol B resulted efficient amplification (protocol A-0%, protocol B-93%, protocol C-13.33%, P <0.05). In a proof-of-principle experiment, the TP53 gene was knocked out in GMECs by CRISPR/Cas9-mediated deletion while constructs containing the anti-VEGF monoclonal antibody (pBC-anti-VEGF) and bacterial L-Asparaginase (pBC-ASNase) transgenes were knocked-in separately in fibroblasts. Detection of successful editing was performed using protocol B and PCR. The integration rates of the pBC-ASNase and pBC-anti-VEGF transgenes were 93.6% and 72%, respectively, as per PCR. The efficiency of biallelic editing in GMECs using CRISPR/Cas9 for the TP53 deletion was 5.4%. Our results suggest that protocol B (heat denaturation/proteinase K) can be used as an inexpensive and quick methodology for detecting genetic modifications in different types of primary goat cells, with efficiency rates consistent with values previously described in the literature when using extraction kits or more complex proteinase K formulations.

Asunto(s)

Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/genética , Análisis Costo-Beneficio , ADN/genética , ADN/aislamiento & purificación , Edición Génica , Transgenes/genética , Animales , Secuencia de Bases , Fibroblastos/citología , Fibroblastos/metabolismo , Cabras

RESUMEN

BACKGROUND: Piercing/sucking insect pests in the order Hemiptera causes substantial crop losses by removing photoassimilates and transmitting viruses to their host plants. Cloning and heterologous expression of plantderived insect resistance genes is a promising approach to control aphids and other sap-sucking insect pests. While expression from the constitutive 35S promoter provides broad protection, the phloem-specific rolC promoter provides better defense against sap sucking insects. The selection of plant-derived insect resistance genes for expression in crop species will minimize bio-safety concerns. RESULTS: Pinellia ternata leaf agglutinin gene (pta), encodes an insecticidal lectin, was isolated and cloned under the 35S and rolC promoters in the pGA482 plant transformation vector for Agrobacterium-mediated tobacco transformation. Integration and expression of the transgene was validated by Southern blotting and qRT-PCR, respectively. Insect bioassays data of transgenic tobacco plants showed that expression of pta under rolC promoter caused 100% aphid mortality and reduced aphid fecundity up to 70% in transgenic tobacco line LRP9. These results highlight the better effectivity of pta under rolC promoter to control phloem feeders, aphids. CONCLUSIONS: These findings suggested the potential of PTA against aphids and other sap sucking insect pests. Evaluation of gene in tobacco under two different promoters; 35S constitutive promoter and rolC phloemspecific promoter could be successfully use for other crop plants particularly in cotton. Development of transgenic cotton plants using plant-derived insecticidal, PTA, would be key step towards commercialization of environmentally safe insect-resistant crops.

Asunto(s)

Áfidos/patogenicidad , Control Biológico de Vectores , Pinellia/química , Virus de Plantas , Nicotiana , Southern Blotting , Reacción en Cadena de la Polimerasa , Regiones Promotoras Genéticas , Plantas Modificadas Genéticamente , Hojas de la Planta/química , Transgenes , Resistencia a la Enfermedad , Protección de Cultivos

RESUMEN

Currently, the generation of cell lines for the production of recombinant proteins has the limitation of unstable gene expression due to the repeat-induced gene silencing or the loss of transgene copies resulting from recombination events. In this work, we developed a new strategy based on the sequential insertion of transgenes for generating stable clones producing high levels of a chimeric human follicle-stimulating hormone (hscFSH). Gene insertion was done by transducing HEK-293 cells with a lentiviral vector containing a bicistronic transcriptional unit for expressing hscFSH and GFP genes. Clone selection was performed by flow cytometry coupled to cell sorting, and the GFP gene was further removed by CRE-mediated site-specific recombination. High-producing clones of hscFSH were obtained after three rounds of lentiviral transduction. Expression levels increased in a step-wise manner from 7 to 23 pg/cell/day, with a relatively constant rate of 7 pg/cell/day in each round of transduction. The GFP gene was successfully removed from the cell genome without disturbing the hscFSH gene expression. Clones generated using this approach showed stable expression levels for more than two years. This is the first report describing the sequential insertion of transgenes as an alternative for increasing the expression levels of transformed cell lines. The methodology described here could notably impact on biotechnological industry by improving the capacity of mammalian cells to produce biopharmaceuticals.

Asunto(s)

Hormona Folículo Estimulante/biosíntesis , Mutagénesis Insercional/métodos , Transgenes/genética , Biotecnología/métodos , Células Clonales , Citometría de Flujo/métodos , Hormona Folículo Estimulante/genética , Expresión Génica , Vectores Genéticos , Proteínas Fluorescentes Verdes/genética , Células HEK293 , Humanos , Lentivirus/genética , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética , Transducción Genética

RESUMEN

Chloroplast transformation has many potential advantages for the production of recombinant proteins in plants. However, it has been reported that chloroplast expression of many proteins, such as human epidermal growth factor (hEGF), results hindered by post-transcriptional mechanisms. hEGF degradation has been related to the redox potential of the stroma and protein misfolding. To solve this problem, we proposed the redirection of hEGF into the thylakoid lumen where the environment could improve disulfide bonds formation stabilizing the functional conformation of the protein. We generated transplastomic tobacco plants targeting hEGF protein to the thylakoid lumen by adding a transit peptide (Str). Following this approach, we could detect thylakoid lumen-targeted hEGF by western blotting while stromal accumulation of hEGF remained undetectable. Southern blot analysis confirmed the integration of the transgene through homologous recombination into the plastome. Northern blot analysis showed similar levels of egf transcripts in the EGF and StrEGF lines. These results suggest that higher stability of the hEGF peptide in the thylakoid lumen is the primary cause of the increased accumulation of the recombinant protein observed in StrEGF lines. They also highlight the necessity of exploring different sub-organellar destinations to improve the accumulation levels of a specific recombinant protein in plastids.

Asunto(s)

Cloroplastos/metabolismo , Factor de Crecimiento Epidérmico/metabolismo , Nicotiana/metabolismo , Plantas Modificadas Genéticamente/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Tilacoides/metabolismo , Transgenes , Cloroplastos/genética , Factor de Crecimiento Epidérmico/genética , Humanos , Plantas Modificadas Genéticamente/genética , Plantas Modificadas Genéticamente/crecimiento & desarrollo , Plastidios/genética , Plastidios/metabolismo , Proteínas Recombinantes de Fusión/genética , Tilacoides/genética , Nicotiana/genética , Nicotiana/crecimiento & desarrollo