Transgene Copy Research Articles

Identification of Loci Enabling Stable and High-Level Heterologous Gene Expression.

Efficient and reliable genome engineering technologies have yet to be developed for diatoms. The delivery of DNA in diatoms results in the random integration of multiple copies, quite often leading to heterogeneous gene activity, as well as host instability. Transgenic diatoms are generally selected on the basis of transgene expression or high enzyme activity, without consideration of the copy number or the integration locus. Here, we propose an integrated pipeline for the diatom, Phaeodactylum tricornutum, that accurately quantifies transgene activity using a β-glucuronidase assay and the number of transgene copies integrated into the genome through Droplet Digital PCR (ddPCR). An exhaustive and systematic analysis performed on 93 strains indicated that 42% of them exhibited high β-glucuronidase activity. Though most were attributed to high transgene copy numbers, we succeeded in isolating single-copy clones, as well as sequencing the integration loci. In addition to demonstrating the impact of the genomic integration site on gene activity, this study identifies integration sites for stable transgene expression in Phaeodactylum tricornutum.

Transgenic mice Cre-dependently expressing mutant polymerase-gamma: novel test-system for pharmacological study of mitoprotective drugs

Introduction: PolG-alpha is a nuclear-encoded enzyme which provides replication and repair of mitochondrial DNA. D257A mutation of PolG-alpha leads to change in the N-terminal ”proofreading” domain, which deprives the enzyme of 3′-5′ exonuclease activity, resulting in accumulation of mutations in the mitochondrial genome.
 Materials and methods: Murine zygotes were microinjected with transgene construction carrying mutant murine Polg coding sequence and GFP coding sequence by a loxP-flanked STOP-cassette. Two Cre-activator strains, CMV-Cre (systemic activation) and Tie2-Cre (endothelial activation), were used for activation of the transgene. To confirm the insertion and Cre-dependent activation of the transgene, genotyping and qPCR copy number measurement of mutant Polg were performed, and GFP fluorescence was assessed.
 Results: Two primary transgenic animals were used as the founders for two lines with copy numbers of transgene ~7 and ~5. After systemic activation, the number of the transgene copies decreases to ~1.0 while endothelial specific activation does not affect the number of transgene copies in tail tissue.
 Discussion: A murine model with spatial control of mutant Polgexpression has been developed. To our knowledge, this is the first transgenic model of tissue-specific mitochondrial dysfunction.
 Conclusion: Transgenic mice Cre-dependent expressing mutant polymerase-gamma are a novel test-system for studying mitochondrial biology and efficacy of mitoprotective drugs.

MAP30 transgenic tobacco lines: from silencing to inducing.

DNA methylation is one of the most important epigenetic event that regulates gene expression. In addition to DNA methylation, transgene copy number may induce gene silencing. Therefore, the study of these cases is useful for understanding of gene silencing regulation. In this study, the methylation pattern of 35S promoter was investigated in the second generation of MAP30 transgenic tobacco lines. Therefore, the genomic DNA melting curve changes were investigated before and after bisulfite treatment by real time PCR. To determine the exact position of methylation, the samples were sequenced after bisulfite treatment. Observation of decrease in DNA melting curve of expressing line in comparison with silenced line confirmed the presence of DNA methylation in silenced line. In order to induce the MAP30 expression, the silenced line was treated using different concentrations of Azacytidine and green tea extracts. The results showed that all concentrations of green tea extracts for 6days and the concentrations of 3 and 10μM Azacytidine for 10 and 3days could induce the expression of MAP30 in silenced line respectively. Finally, the transgene copy number was estimated using real time PCR, as silenced line contained more than two copies while the lines expressing MAP30 contained only one or two copies. Finally, we found that the presence of DNA methylation and also multiple gene copy numbers in silenced line have been led to gene silencing. Moreover, the effect of green tea extract on DNA methylation showed incredible results for the first time.

An efficient sorghum transformation system using embryogenic calli derived from mature seeds.

Significant progress has been made on sorghum transformation in the last decades; however, the transformation process has been constrained by the availability of immature embryos because most of the researchers have utilized immature embryos as favorable explants. Although immature embryos have been proven to be optimal for tissue culture and transformation, isolation of immature embryos is time-consuming, labor-intensive, and limited by warm weather. In this study, we developed an efficient genetic transformation system using mature seeds as explants. The nptII and gus gene, used as the selective marker and report gene respectively, have been co-transformed by particle bombardment. After optimization of tissue culture, the G418 concentration, and transgenic, the average transformation frequency at 13.33% was achieved routinely. The transgenic events and transgene copy numbers were determined by PCR and RT-PCR, respectively. The geneticin selection and GUS staining on T1 seedlings confirmed that the transgenic plants were heritable. Our results demonstrated that the efficient sorghum transformation system has been established using mature seeds as explants. This transformation system will promote sorghum research on genetic engineering and genome editing without seasonal weather conditions restriction and explant resources restriction.

Targeted T cell receptor gene editing provides predictable T cell product function for immunotherapy

SummaryAdoptive transfer of T cells expressing a transgenic T cell receptor (TCR) has the potential to revolutionize immunotherapy of infectious diseases and cancer. However, the generation of defined TCR-transgenic T cell medicinal products with predictable in vivo function still poses a major challenge and limits broader and more successful application of this “living drug.” Here, by studying 51 different TCRs, we show that conventional genetic engineering by viral transduction leads to variable TCR expression and functionality as a result of variable transgene copy numbers and untargeted transgene integration. In contrast, CRISPR/Cas9-mediated TCR replacement enables defined, targeted TCR transgene insertion into the TCR gene locus. Thereby, T cell products display more homogeneous TCR expression similar to physiological T cells. Importantly, increased T cell product homogeneity after targeted TCR gene editing correlates with predictable in vivo T cell responses, which represents a crucial aspect for clinical application in adoptive T cell immunotherapy.

Abstract 509: Conversion of cellular kinetic data for chimeric antigen receptor T-cell therapy (CAR-T) into interpretable units

Abstract Objectives: Cellular kinetic (CK) measurement of CAR-T cell expansion in-vivo by quantitative polymerase chain reaction (qPCR) has been measured in units of transgene copy number/μg of DNA. We propose a formula to convert the qPCR, and flow cytometry (FC) measurements to interpretable numbers of CAR T-cells/μL blood. Further, after the conversion CK parameters were correlated with efficacy/safety endpoints. Methods: CK data measured using both qPCR and FC assays were utilized from the ELIANA trial in pediatric and young adult patients with relapsed and refractory acute lymphoblastic leukemia (pALL). qPCR measures the presence of CAR transgene in cells with units of copies of CAR-DNA/μg of genomic DNA, while FC quantifies surface expression of CAR T-cells as the % of either T-cells or white blood cells (WBCs) that express CAR. Neither measurement accounts for the typical significant increase in WBCs following CAR-T infusion. We propose equations for converting FC (Eq1) or qPCR (Eq2) into concentration of CAR-cells/μL of blood, using WBC counts from the complete blood count. For qPCR, the equation relies on 3 additional parameters M·F/N: amount of DNA/WBC (M), average number of copies of CAR-DNA/CTL019 cell (N), and fraction of cells with CAR-DNA that express CAR receptor (F). To estimate M·F/N, we performed regression of the cells/μL estimate from FC vs qPCR. We fit the CK model [1] to obtain the model parameters. Finally, CK parameters with safety/efficacy were correlated to compare the converted estimates to the native CK units. • (CD3+CAR+cells)/μl blood= WBC/μl blood × (CD3+CAR+cells)/WBC (1) • (CAR+cells)/μl blood=WBC/μl blood × CAR DNA copies/μg DNA × M μg DNA/WBC × 1 CAR cell/N CAR DNA copies×F (2) Results: There was high correlation between FC and qPCR estimates of CAR-T in the blood of pALL patients (r2=0.775). The M·F/N value derived based on FC and qPCR results was estimated as 2.68e-6μg DNA/CAR copies. Assuming M=6.6e-6μg DNA/WBC [2] and F=1, this predicts that N=2.46 CAR copies/CAR-T cell. There was also high correlation between the copies/μg and cells/μL estimates using qPCR (r2=0.752). The relationship between CK parameters and safety/efficacy endpoints was not improved when cells/μL was used. This can be attributed to high correlation between these metrics. Using the CK model, 11x greater fold expansion was predicted using the cells/μL estimate compared to copies/μg. This is because cells/μL estimate accounts the expansion of both CAR-T and WBC numbers following lymphodepletion. Conclusions: The conversion of CK into cells/μL allows for a physiological interpretation of CK data with a high correlation between the cells/μL and copies/μg indicating either metric can be used to predict safety/efficacy.

Phase I/II Study of Pembrolizumab for Progressive Diffuse Large B Cell Lymphoma after Anti-CD19 Directed Chimeric Antigen Receptor Modified T Cell Therapy

Phase I/II Study of Pembrolizumab for Progressive Diffuse Large B Cell Lymphoma after Anti-CD19 Directed Chimeric Antigen Receptor Modified T Cell Therapy

New homozygous gpt delta transgenic rat strain improves an efficiency of the in vivo mutagenicity assay

BackgroundGene mutation assays in transgenic rodents are useful tools to investigate in vivo mutagenicity in a target tissue. Using a lambda EG10 transgene containing reporter genes, gpt delta transgenic mice and rats have been developed to detect point mutations and deletions. The transgene is integrated in the genome and can be rescued through an in vitro packaging reaction. However, the packaging efficiency is lower in gpt delta rats than in mice, because of the transgene in gpt delta rats being heterozygous and in low copy number. To improve the packaging efficiency, we herein describe a newly developed homozygous gpt delta rat strain.ResultsThe new gpt delta rat has a Wistar Hannover background and has been successfully maintained as homozygous for the transgene. The packaging efficiency in the liver was 4 to 8 times higher than that of existing heterozygous F344 gpt delta rats. The frequency of gpt point mutations significantly increased in the liver and bone marrow of N-nitroso-N-ethylurea (ENU)- and benzo[a]pyrene (BaP)-treated rats. Spi− deletion frequencies significantly increased in the liver and bone marrow of BaP-treated rats but not in ENU-treated rats. Whole genome sequencing analysis identified ≥ 30 copies of lambda EG10 transgenes integrated in rat chromosome 1.ConclusionsThe new homozygous gpt delta rat strain showed a higher packaging efficiency, and could be useful for in vivo gene mutation assays in rats.

Transgene behavior in genetically modified teosinte hybrid plants: transcriptome expression, insecticidal protein production and bioactivity against a target insect pest

BackgroundIn 2009, Spanish farmers reported a novel weed, now identified as a relative of maize’s ancestor, teosinte, in their maize fields. Introgression of the insect resistance transgene cry1Ab from genetically modified (GM) maize into populations of this weedy Spanish teosinte could endow it with additional defense mechanisms. The aims of this study were: (1) to test if hybridization between GM maize and weedy plants from Spain is possible; (2) to understand the relationship between transgene transcription activity, concentrations of the expected transgene product (Cry1Ab protein) and the bioactivity of the latter on target insect pests following transgene flow from GM maize into Spanish teosinte plants.ResultsWe demonstrated that hybridization between GM maize and the weedy Spanish teosinte is possible, with no observable barrier to the formation of crop/weed hybrids when teosinte served as pollen donor. When GM maize plants were used as pollen donors, significant crossing incompatibility was observed: hybrid plants produced only few “normal” seeds. Nevertheless, viable F1 seeds from GM pollen crossed onto teosinte were indeed obtained. The cry1Ab transgene was stably expressed as mRNA in all crossings and backgrounds. Similarly, toxicity on neonate Ostrinia nubilalis, presumably due to Cry1Ab protein, was consistently expressed in teosinte hybrids, with mortality rates 95% or higher after only 4 days exposure, similar to rates on parental GM maize plants. Nevertheless, no strong correlations were observed between transgene transcription levels and Cry1Ab concentrations, nor between Cry1Ab concentrations and insect mortality rates across all of the different genetic backgrounds.ConclusionsOur results establish fundamental parameters for environmental risk assessments in the European context: first, we show that crop/weed hybridization in fields where maize and teosinte exist sympatrically can lead to potentially catastrophic transfer of resistance traits into an already noxious weed; second, our results question the viability of using gene dosage to model and predict ecological performance in either the intended crop plant or the undesired teosinte weed. Significant questions remain that should be addressed in order to provide a scientific, sound approach to the management of this novel weed.

LINE-1 vectors mediate recombinant antibody gene transfer by retrotransposition in Chinese hamster ovary cells.

Retrotransposons, such as long interspersed element-1 (LINE-1), can copy themselves to other genomic loci via a transposition event (termed retrotransposition). Retrotransposons, therefore, have potential use as an efficient gene delivery tool to integrate multiple copies of a target gene into a host genome. Here, we developed a retrotransposon vector based on LINE-1 that achieves target gene integration of multiple transgene copies. The retrotransposon vector contains a neomycin resistance gene split by an intron as a marker gene, and a gene encoding an antibody single-chain variable fragment (Fv) fused with the constant antibody region (Fc) (scFv-Fc) as a model target gene. G418-resistant Chinese hamster ovary cells were generated using this retrotransposon vector, and scFv-Fc was produced in the culture medium. To regulate retrotransposition, we developed a retrotransposon vector system that separately expressed the two open reading frames (ORF1 and ORF2) of LINE-1. Genomic PCR analysis detected the transgene sequence in almost all tested clones. Compared with clones established using the intact LINE-1 vector, clones generated with the split ORF1 and ORF2 system showed similar specific scFv-Fc productivity and retrotransposition efficiency. This approach of using a retrotransposon-based vector system has the potential to provide a new gene delivery tool for mammalian cells.

Phase 1 Study of CART-ddBCMA, a CAR-T therapy utilizing a novel synthetic binding domain, for the treatment of subjects with relapsed and refractory multiple myeloma.

8015 Background: CART-ddBCMA is an autologous CAR-T cell therapy encoding a novel non-scFv synthetic binding domain targeting BCMA with a 4-1BB costimulatory motif and CD3-zeta T-cell activation domain. The novel binding domain is based on a computationally-derived triple-helix protein scaffold that is small (73 amino acids), stable, engineered to reduce immunogenicity, and can be modified to bind alternative targets. Methods: ARC-101 (NCT04155749), ARM 1 (CART-ddBCMA) is a Phase 1, multi-center, open-label, dose escalation trial enrolling subjects who have received ≥3 prior regimens, including proteasome inhibitor(s), immuno-modulatory agent(s), and anti-CD38 antibody, or are triple-refractory. Subjects undergo lymphodepletion with fludarabine and cyclophosphamide, then receive CART-ddBCMA as a single infusion. Planned dose levels are 100, 300, and up to 900 x 106 CAR+ T cells. The primary endpoint is incidence of adverse events (AEs), including dose-limiting toxicities (DLTs). Secondary endpoints include clinical response per IMWG criteria, MRD, DOR, PFS, OS, and CAR-T cell kinetics. Results: As of 29 Jan 2021, 10 subjects received CART-ddBCMA, 9 subjects were evaluable, and 1 subject was pending assessment. Median age was 66 years [min:max 54 to 75]. 6 subjects received 100 x 106 CAR+ T cells, and 4 subjects received 300 x 106 CAR+ T cells. Median CAR+ expression was 74.5% (min:max 61-87%) of total T cells. Of the evaluable subjects, median follow-up after cell infusion was 208 days (min:max 45 to 355+ days), 9/9 subjects were penta-refractory, 1 subject was also refractory to BCMA-directed ADC. 8/9 had high-risk cytogenetics (1 subject’s sample not evaluable), and 6/9 subjects had extramedullary disease. No DLTs were reported. Per ASTCT Consensus Grading (Lee et al, 2019), 8 subjects developed G1/2 CRS, 1 subject in the higher dose cohort developed G3 CRS that rapidly resolved with tocilizumab. 1 subject developed G2 ICANS which rapidly resolved with intervention. 7 subjects received tocilizumab; 3 received dexamethasone. ORR was 100% (9/9) per IMWG criteria including 4 sCR, 1 VGPR, and 4 PR. 1 subject with PR relapsed and was retreated. All other subjects have ongoing responses; observations included sFLC normalization and elimination of detectable bone marrow disease by Month 1. Ongoing responses for subjects not yet achieving CR continue to deepen. 7 subjects were evaluable by MRD of which 5 are MRD-negative, and 2 are pending results. CAR-T cell expansion, as measured by vector transgene copies per microgram genomic DNA was observed in all patients. Conclusions: Early efficacy results are encouraging, with 9/9 (100%) ORR and manageable toxicities. 8/9 responses are ongoing and responses continue to deepen. These data are encouraging in high-risk subjects with penta-refractory myeloma. Subjects continue to be enrolled and treated. Clinical trial information: NCT04155749.

EPO transgene detection in dried blood spots for antidoping application

The modification of gene expression to treat diseases is a field of research with exponential growth. As doping in sport closely follows emerging therapies, a surveillance of the modification of gene expression to enhance performance is needed. The gene coding for erythropoietin (EPO) is one target of interest. Since 2010, several protocols have been proposed to identify EPO gene doping by focusing on the presence in blood of a transgene that differ in size from the endogenous gene sequence, normally found in the human DNA. In this work, our aim was to validate an easily applicable method for EPO gene doping detection in dried blood spots (DBS). We evaluated the detection of EPO transgene in 20-μl DBS after the spike of a plasmid carrying the EPO transgene in whole blood. Three different DBS were compared: Nucleic-Card™, Whatman® 903, and the volumetric 20-μl VAMS™. Detection was performed with real-time polymerase chain reaction (PCR) and validated with two Taqman assays (one commercial and one custom) specific for the EPO transgene. The initial testing procedure could be done using one assay (custom) and the confirmation using the second one (commercial Taqman) with a final check of the size of the PCR product. Starting from 20-μl dried blood, 1000 copies of EPO transgene could efficiently be detected with the three types of DBS, VAMS showing a slightly better sensitivity. No loss of sensitivity was observed after 1-month storage of DBS at room temperature. This method could be applied to DBS collected during doping controls and allows reanalysis.

Investigation of product-derived lymphoma following infusion of piggyBac-modified CD19 chimeric antigen receptor T cells

AbstractWe performed a phase 1 clinical trial to evaluate outcomes in patients receiving donor-derived CD19-specific chimeric antigen receptor (CAR) T cells for B-cell malignancy that relapsed or persisted after matched related allogeneic hemopoietic stem cell transplant. To overcome the cost and transgene-capacity limitations of traditional viral vectors, CAR T cells were produced using the piggyBac transposon system of genetic modification. Following CAR T-cell infusion, 1 patient developed a gradually enlarging retroperitoneal tumor due to a CAR-expressing CD4+ T-cell lymphoma. Screening of other patients led to the detection, in an asymptomatic patient, of a second CAR T-cell tumor in thoracic para-aortic lymph nodes. Analysis of the first lymphoma showed a high transgene copy number, but no insertion into typical oncogenes. There were also structural changes such as altered genomic copy number and point mutations unrelated to the insertion sites. Transcriptome analysis showed transgene promoter–driven upregulation of transcription of surrounding regions despite insulator sequences surrounding the transgene. However, marked global changes in transcription predominantly correlated with gene copy number rather than insertion sites. In both patients, the CAR T-cell–derived lymphoma progressed and 1 patient died. We describe the first 2 cases of malignant lymphoma derived from CAR gene–modified T cells. Although CAR T cells have an enviable record of safety to date, our results emphasize the need for caution and regular follow-up of CAR T recipients, especially when novel methods of gene transfer are used to create genetically modified immune therapies. This trial was registered at www.anzctr.org.au as ACTRN12617001579381.

Optimizing expression of a single copy transgene in C. elegans.

The utility of single copy transgenic insertions in C. elegans is often limited by low expression. We examined the effects of modifying the trans-splicing signal, the Kozak ribosome binding site, the N-terminal amino acid of the reporter and the 3′ UTR sequences on the expression level of a mec-4 promoter GFP transgene. The trans-splicing signal and the 3′ UTR had most dramatic effects on expression while modifying the Kozak signal or the N-terminal amino acid had less influence on expression.

Amplicon-based RNAi construct targeting beta-C1 gene gives enhanced resistance against cotton leaf curl disease.

The online version contains supplementary material available at 10.1007/s13205-021-02816-6.

Chimeric antigen receptor T cells targeting CD7 in a child with high-risk T-cell acute lymphoblastic leukemia

Effective systemic treatments for relapsed or refractory T-cell acute lymphoblastic leukemia (T-ALL) are limited. Recent clinical application of chimeric antigen receptor (CAR) immunotherapy has demonstrated successful control of B-cell malignancies by CAR-T cells; however, designing CARs for T-ALL remains a challenge. CD7 overexpression in T-cell malignancies may be an attractive target for immunotherapy in T-ALL. This study aimed to describe the safe and effective use of autologous CD7-CAR T cells (4SCAR7) for the treatment of T-ALL with induction failure in an 11-year-old patient. Based on The Chinese Children’s Cancer Group-ALL (CCCG-ALL) study protocol, minimal residual disease (MRD) by flow cytometry (FC) analysis was detected on days 19 and 46 of remission induction. At the end of remission-induction chemotherapy, the patient achieved morphologic complete remission, though with MRD 16.13% and RT-PCR of KMT2A-MLLT1 fusion positive, which indicated induction failure. The cerebrospinal fluid (CSF) was negative for blasts at diagnosed. CAR-T therapy and allogeneic transplant were recommended as the next treatment options. CD3+ lymphocytes were collected from the patient 18 days after the high-dose MTX chemotherapy through leukapheresis. The 4SCAR7 CD7-targeting CAR-T cells were generated thereafter. The patient received lymphodepleting chemotherapy prior to 4SCAR7 infusion. Oral administration of itraconazole and sulfamethoxazole was performed from day 0 after CAR-T cell infusion. The patient did not have hypotension, hypoxia, or serious biochemical change or abnormality, but had fever on day 9. Although grade 1 cytokine-release syndrome (CRS) was diagnosed, it was successfully treated with ibuprofen. Anti-CD7 CAR transgene copy numbers in peripheral blood were determined by qPCR, which showed effective expansion initially, then dropped quickly, and persisted at a low level. Although experienced cytopenia from days 14 to 21, the patient achieved remission on day 17. After complete remission, the patient received hematopoietic stem cell transplantation (HSCT) and has recovered well to thisdate. Overall, this report suggested that 4SCAR7 could be a safe and effective strategy for the treatment of pediatric patients with high-risk T-cell malignancies.

In Vivo Function of Flow-Responsive Cis-DNA Elements of eNOS Gene: A Role for Chromatin-Based Mechanisms.

eNOS (endothelial nitric oxide synthase) is an endothelial cell (EC)-specific gene predominantly expressed in medium- to large-sized arteries where ECs experience atheroprotective laminar flow with high shear stress. Disturbed flow with lower average shear stress decreases eNOS transcription, which leads to the development of atherosclerosis, especially at bifurcations and curvatures of arteries. This prototypic arterial EC gene contains 2 distinct flow-responsive cis-DNA elements in the promoter, the shear stress response element (SSRE) and the KLF (Krüppel-like factor) element. Previous in vitro studies suggested their positive regulatory functions on flow-induced transcription of EC genes including eNOS. However, the in vivo function of these cis-DNA elements remains unknown. Insertional transgenic mice with a mutation at each flow-responsive cis-DNA element were generated using a murine eNOS promoter-β-galactosidase reporter by linker-scanning mutagenesis and compared with episomal-based mutations in vitro. DNA methylation at the eNOS proximal promoter in mouse ECs was assessed by bisulfite sequencing or pyrosequencing. Wild type mice with a functional eNOS promoter-reporter transgene exhibited reduced endothelial reporter expression in the atheroprone regions of disturbed flow (n=5). It is surprising that the SSRE mutation abrogated reporter expression in ECs and was associated with aberrant hypermethylation at the eNOS proximal promoter (n=7). Reporter gene silencing was independent of transgene copy number and integration position, indicating that the SSRE is a critical cis-element necessary for eNOS transcription in vivo. The KLF mutation demonstrated an integration site-specific decrease in eNOS transcription, again with marked promoter methylation (n=8), suggesting that the SSRE alone is not sufficient for eNOS transcription in vivo. In wild type mice, the native eNOS promoter was significantly hypermethylated in ECs from the atheroprone regions where eNOS expression was markedly repressed by chronic disturbed flow, demonstrating that eNOS expression is regulated by flow-dependent DNA methylation that is region-specific in the arterial endothelium in vivo. We report, for the first time, that the SSRE and KLF elements are critical flow sensors necessary for a transcriptionally permissive, hypomethylated eNOS promoter in ECs under chronic shear stress in vivo. Moreover, eNOS expression is regulated by flow-dependent epigenetic mechanisms, which offers novel mechanistic insight on eNOS gene regulation in atherogenesis.

POS-432 DELETION OF THE POLYCYSTIN-1 COILED-COIL MOTIF LEADS TO A SEVERE FUNCTIONAL HYPOMORPHIC ADPKD ALLELE IN MICE: INSIGHTS INTO INTRACELLULAR MECHANISMS

Autosomal dominant polycystic kidney disease (ADPKD) is the most prevalent genetic disorder that is characterized by the formation of bilateral renal cysts and leads to kidney failure. Majority of ADPKD cases results from mutations in the PKD1 gene encoding polycystin-1/Pc-1. Another gene PKD2 is involved in this disease and encodes polycystin-2/Pc-2. While the functions of both proteins remain to be elucidated, in vitro study has demonstrated that the carboxy terminal regions of Pc-1 and Pc-2 could interact together through their coiled-coil motif. We have deleted the coiled-coil (ΔCC) domain from a Pkd1-BAC by homologous recombination. The purified Pkd1ΔCCfragment served to produce four transgenic mouse lines (1-30 copies of the transgene). These Pkd1ΔCCtransgenic lines where compared to control Pkd1TAG mouse lines (14-16 copies of transgene) that contain a full length wild-type Pkd1. These two series of transgenic lines display identical expression profile to the endogenous gene and the level of expression depends on the number of copies. While Pkd1TAG mice develop PKD in a severity proportional to the level of expression of the transgene, the Pkd1ΔCClines are spared from PKD. These results demonstrate the importance of the coiled-coil motif in the development of the disease. To analyze Pkd1ΔCCtransgene independently from the endogenous gene, the Pkd1ΔCC mice where crossed on a Pkd1-/-genetic background that develop PKD and dies by birth. Interestingly, the resulting Pkd1-/-; Pkd1ΔCCmice survive past birth: these Pkd1-/-; Pkd1ΔCC mice with one copy of the transgene show renal cysts and die at ~ 2 weeks, while the high-copy Pkd1-/-; Pkd1ΔCCmice have no phenotype like the Pkd1TAG mice on a Pkd1-/- background. Because Pkd1+/-mice have no phenotype, these genetic complementation results suggest that Pc1ΔCC is an hypomorphic allele. Biochemical characterization show that Pc1ΔCC undergoes autoproteolytic cleavage at G protein-coupled receptor proteolytic site (GPS)/GAIN domain with similar cleavage efficiency as the native Pc-1. Intracellular transport analysis of Pc1ΔCC revealed a delay in maturation in the ER / Golgi network, consistent with a genetic hypomorph. Pc-1ΔCC secretion into exosomes both in vivo and in primary MEFs is also reduced. While Pc-1 and Pc-2 appear to interact in vitro through their coiled-coil domain and are co-transported, Pc1ΔCC interaction with Pc-2 is conserved in the kidney, suggesting a distinct site of interaction and mechanism in vivo. Altogether, our results show that the coiled-coil motif in vivo is implicated in Pc-1 maturation independently of Pc-2 and highlight the existence of a critical partner in intracellular transport.

Digital PCR (dPCR) and qPCR mediated determination of transgene copy number in the forage legume white clover (Trifolium repens).

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.

Transgenic Expression of dsRNA Targeting the Pentalonia nigronervosa acetylcholinesterase Gene in Banana and Plantain Reduces Aphid Populations.

The banana aphid, Pentalonia nigronervosa, is the sole insect vector of banana bunchy top virus (BBTV), the causal agent of banana bunchy top disease. The aphid acquires and transmits BBTV while feeding on infected banana plants. RNA interference (RNAi) enables the generation of pest and disease-resistant crops; however, its effectiveness relies on the identification of pivotal gene sequences to target and silence. Acetylcholinesterase (AChE) is an essential enzyme responsible for the hydrolytic metabolism of the neurotransmitter acetylcholine in animals. In this study, the AChE gene of the banana aphid was targeted for silencing by RNAi through transgenic expression of AChE dsRNA in banana and plantain plants. The efficacy of dsRNA was first assessed using an artificial feeding assay. In vitro aphid feeding on a diet containing 7.5% sucrose, and sulfate complexes of trace metals supported aphid growth and reproduction. When AChE dsRNA was included in the diet, a dose of 500 ng/μL was lethal to the aphids. Transgenic banana cv. Cavendish Williams and plantain cvs. Gonja Manjaya and Orishele expressing AChE dsRNA were regenerated and assessed for transgene integration and copy number. When aphids were maintained on elite transgenic events, there was a 67.8%, 46.7%, and 75.6% reduction in aphid populations growing on Cavendish Williams, Gonja Manjaya, and Orishele cultivars, respectively, compared to those raised on nontransgenic control plants. These results suggest that RNAi targeting an essential aphid gene could be a useful means of reducing both aphid infestation and potentially the spread of the disease they transmit.