Transgene Copy Research Articles

Transgene behavior in Zea mays L. crosses across different genetic backgrounds: Segregation patterns, cry1Ab transgene expression, insecticidal protein concentration and bioactivity against insect pests.

Brazil and South Africa, countries with economies in transition, are characterized by a dual agrarian structure co-occurring, sometimes, alongside in the same region. Large-scale commercial farming produces crops for export to global markets on the one hand, and small-scale farming, on the other hand, provides for subsistence and local markets. In both systems, maize (Zea mays) is a key crop for these two countries. For the commercial system, maize is a commodity crop while for the small-scale system it is the prime staple crop. In commercial systems, farmers predominantly grow genetically modified (GM) hybrid maize. In small-scale systems, farmers rely on open pollinated varieties (OPVs) and/or landraces and are largely dependent on seed saving systems. The aim of this study was to understand the relationship between transgene expression rates, the resulting concentrations of the transgene product (Bt protein) and its bioactivity in insect pests following transgene flow from GM hybrid maize into non-genetically modified, non-GM near-isogenic maize hybrid (ISO) and OPVs. We modeled segregation patterns and measured cry1Ab transgene expression (mRNA quantification), Cry1Ab protein concentration and pest survival. Two groups of F1, F2 crosses and backcrosses with GM, ISO and OPV maize varieties from Brazil and South Africa were used. Bioassays with the larvae of two lepidopteran maize pest species, Helicoverpa armigera and Spodoptera littoralis, were carried out. Overall, the cry1Ab transgene outcrossed effectively into the genetic backgrounds tested. The cry1Ab transgene was stably expressed in both ISO and OPV genetic backgrounds. Transgene introgression led to consistent, though highly variable, concentrations of Cry1Ab toxins that were similar to those observed in GM parental maize. Most crosses, but not all, suggested the expected Mendelian segregation pattern. Transgene expression rates were significantly higher than expected from purely Mendelian segregation in the South African crosses. In South African materials, ISO and OPV crosses produced significantly lower Cry1Ab concentrations compared to the GM parental maize. The Cry1Ab toxins from crosses were bioactive and induced mortality rates of ≥92.19% in H. armigera and ≥40.63% in S. littoralis after a period of only 4 days. However, no correlations were observed between the quantitation of mRNA for cry1Ab and the corresponding Cry1Ab protein concentrations, nor between the Cry1Ab concentrations and insect mortality rates across different genetic backgrounds. We therefore suggest that while transcription of the cry1Ab transgene reliably determines the presence of Cry1Ab protein, mRNA levels do not reflect, by themselves, the end Cry1Ab protein concentrations found in the plant. Because predictably high Cry1Ab concentrations are a key prerequisite for effective insect resistance management (IRM) programs, this observation raises questions about the effectiveness of such programs in scenarios with complex crop genetic backgrounds. On the other hand, confirmed bioactivity in all crosses should be expected to impact small farmer’s selection behavior, unknowingly favoring the insecticidal trait. This may lead to a fixation of the trait in the wider population, and may influence the genetic diversity of varieties maintained by small-scale farmers.

A simple quantitative PCR assay to determine TRAMP transgene zygosity

BACKGROUND.The TRansgenic Adenocarcinoma of the Mouse Prostate (TRAMP) model remains one of the most widely used transgenic mouse models of prostate cancer. This is due to its ability to recapitulate with ~100% penetrance multiple aspects of the human disease such as prostatic intraepithelial neoplasia lesions, invasive carcinoma, progression to castration-resistant prostate cancer including aggressive neuroendocrine prostate cancer and metastasis. Despite its popularity, the use of TRAMP mice is limited/slowed by the inability to distinguish the zygosity of the TRAMP transgene. This is especially true for breeding strategies implementing multiple crosses and alleles and when the rapid generation of large animal cohorts with the desired genotype is needed.METHODS.We developed a quantitative PCR (qPCR) approach to determine the relative TRAMP transgene copy number of mice.RESULTS.This method was validated by three independent laboratories across two institutions, which successfully identified the genotype of the mice 98.2% of the time (165/168) in the first attempt. The genotypes of the uncertain mice were correctly identified in the repeated experiments.CONCLUSIONS.We develop the first straightforward, quantitative PCR (qPCR) approach to reliably determine the TRAMP transgene zygosity. The development of this qPCR-based genotyping method enables researchers to streamline breeding strategies when creating complex genetic mouse models involving TRAMP mice; thus, ultimately reducing the required animal numbers, cost, and investigator time.

The Whys and Wherefores of Transitivity in Plants.

Transitivity in plants is a mechanism that produces secondary small interfering RNAs (siRNAs) from a transcript targeted by primary small RNAs (sRNAs). It expands the silencing signal to additional sequences of the transcript. The process requires RNA-dependent RNA polymerases (RDRs), which convert single-stranded RNA targets into a double-stranded (ds) RNA, the precursor of siRNAs and is critical for effective and amplified responses to virus infection. It is also important for the production of endogenous secondary siRNAs, such as phased siRNAs (phasiRNAs), which regulate several genes involved in development and adaptation. Transitivity on endogenous transcripts is very specific, utilizing special primary sRNAs, such as miRNAs with unique features, and particular ARGONAUTEs. In contrast, transitivity on transgene and virus (exogenous) transcripts is more generic. This dichotomy of responses implies the existence of a mechanism that differentiates self from non-self targets. In this work, we examine the possible mechanistic process behind the dichotomy and the intriguing counter-intuitive directionality of transitive sequence-spread in plants.

Fusion with matrix attachment regions enhances expression of recombinant protein in human HT-1080 cells

Like endogenous proteins, recombinant foreign proteins produced in human cell lines also need post-translational modifications. However, high and long-term expression of a gene of interest (GOI) presents significant challenges for recombinant protein production in human cells. In this work, the effect of human matrix attachment region elements (MARs), including the β-globin MAR (gMAR), chicken lysozyme MAR (cMAR), and a combination of these two, on the stable expression of GOI was assessed in human HT-1080cells. After transfection with vectors containing the MAR elements and eGFP, stably HT-1080cell pools were obtained under selective pressure. eGFP protein expression was analyzed by flow cytometry, while transgene copy number and eGFP mRNA expression levels were determined with qPCR and qRT-PCR technology. We found that MARs could not enhance transfection efficiency, but gMAR could significantly increase eGFP expression in stable HT-1080cell pools by approximately 2.69-fold. Moreover, gMAR could also increase eGFP expression stability during long-term culture. Lastly, we showed that the effect of the MARs on transgenes was related to the gene copy number. In summary, this study found that MARs could both enhance the transgene expression and stability in HT-1080cells.

Fluorescence-assisted sequential insertion of transgenes (FASIT): an approach for increasing specific productivity in mammalian cells

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.

Lack of methylation on transgene leads to high level and persistent transgene expression in induced pluripotent stem cells

Short-lived therapeutic gene expression in mammalian cells by DNA methylation is one of the major challenges in gene therapy. In this study, we assessed the implication of DNA methylation on the duration of GFP expression in mouse embryonic stem (ES) and mouse induced pluripotent stem (iPS) cells. The cells were transduced with lentivirus (LV) carrying green fluorescent protein (GFP) driven by either human elongation factor (EF1α) or cytomegalovirus (CMV) promoter. Transduced iPS cells exhibited higher percentage of GFP+ cells with persistent mean fluorescent intensity than transduced ES cells. Analysis on the integrated copy of transgene in the population of the transduced cells demonstrated similar copy number. However, significant increase in GFP intensity following 5-azaC treatment was observed in transduced ES cells only, suggesting the influence of DNA methylation in transgene silencing. Subsequent DNA methylation analysis showed that the promoter and the GFP region of the provirus in iPS cells had negligible methylation profile compared to transduced ES cells. Interestingly, sustained transgene expression was observed upon directed differentiation of transduced iPS cells towards CD34+ CD45+ cells. Hence, this study has shown that favourable transgene activity from lentiviral transduced iPS cells was due to the lack of methylation at the proviral regions.

Analysis of IL-6 serum levels and CAR T cell-specific digital PCR in the context of cytokine release syndrome

Chimeric antigen receptor T-cell (CAR-T) therapies are increasingly used to treat relapsed B-cell lymphomas and acute lymphoblastic leukemia. Considering the frequency of cytokine release syndrome and CAR-T-related encephalopathy syndrome (CRS/CRES) after CAR-T administration, strategies enabling timely prediction of impending CRS/CRES are a clinical need. We evaluated the dynamics of serum interleukin (IL)-6 levels and CAR-T transgene copy numbers by digital droplet polymerase chain reaction in the peripheral blood of 11 consecutive patients with aggressive B-cell malignancies. Four of 11 patients developed CRS, and 3 patients had CRES (33%), 2 of them had previous CRS. IL-6 levels increased on the day of clinical manifestation of CRS. All CRS patients had increased IL-6 peak levels (median IL-6 peak 606 pg/mL in CRS patients vs. 22 pg/mL in non-CRS patients, p = 0.0061). Different patterns emerged from the dynamics of CAR-T/µg genomic DNA: "rapid increase and rapid decrease with complete disappearance," "rapid increase and slow decrease with higher persistence," "rapid increase and rapid decrease with lower persistence," and "slow increase and rapid decrease with almost disappearance." Patients with the pattern "rapid increase and slow decrease with higher persistence" of CAR-T/µg genomic DNA concentration seemed to be at higher risk of developing CRS/CRES. Thus, the dynamics of CAR-T transgene copy numbers merits further evaluation for a possible association with manifestation of CRS. Increased IL-6 serum levels at CRS manifestation may contribute to the interpretation of symptoms.

Poly (ADP-ribose) polymerase inhibitor 3-methoxybenzamide enhances in vitro plant growth, microtuberization, and transformation efficiency of blue potato (Solanum tuberosum L. subsp. andigenum)

Poly(ADP-ribose) polymerases (PARP) are involved in the regulation of plant growth and development, especially under stress conditions. These enzymes post-translationally modify target proteins by adding ADP-ribose polymers. This study presents the effect of PARP chemical inhibitor 3-methoxybenzamide (3 MB) on in vitro plant growth, microtuber formation, and Agrobacterium-mediated transformation of blue potato (Solanum tuberosum L. subsp. andigenum). The addition of 3 MB (0.2 to 0.6 mM) significantly increased the growth and microtuber formation of in vitro propagated plants. The highest gain in plant fresh weight, root mass, and microtuberization was observed in clonal propagation medium with 0.4 mM 3 MB. Transformation frequency of blue potato internodes increased to 43%, 2.7-fold over the non-treated control (16%), when 0.2 mM 3 MB was included in the culture medium during the whole process. The addition of 3 MB during Agrobacterium transformation did not affect transgene copy number in regenerated plants. Southern blot analyses and histochemical staining for s-glucuronidase activity confirmed the presence and expression, respectively, of the uidA transgene in plant tissues. From these results, it can be concluded that the inhibition of PARP activity can increase biomass production and transformation rates for genetic improvement of blue potato.

Low-temperature pretreatment of explants and high maltose concentration during callus culture improves particle-bombardment-mediated stable transgene expression in common wheat.

Biolistic transformation systems are widely used to introduce foreign genes into common wheat (Triticum aestivum L.); however, these techniques often generate high transgene copy numbers and complex transgene integration patterns that hinder the stable expression of the transgenes. To improve the efficiency of stable transgene expression, we examined the effect of low-temperature pretreatment of wheat flower spikes and of high maltose concentration (HMC) in the medium during the subsequent callus culture. Tillers of the spring wheat cultivar Bobwhite were stored at 5°C without water for one week before the isolation of their immature scutellar tissues, and the resulting particle-bombarded explants were cultured on 15% maltose for a month. Together, these treatments significantly increased the number of recovered transgenic lines expressing the reporter gene. The low-temperature pretreatment eliminated the negative effects of HMC, and HMC improved the efficiency of stable transgene expression. Southern blot analysis revealed that transgenic lines recovered after HMC treatment integrated a lower copy number of transgenes than those cultured at normal (4%) maltose concentration. These findings suggest that the HMC-mediated reduction of the transgene copy number results from the suppression of plasmid DNA rearrangement before or during transgene integration into the wheat genome.

Expression of cry1Aa gene in cabbage imparts resistance against diamondback moth (Plutella xylostella).

Brassica oleracea cv. Pride of India is one of the most promising vegetable cultivars commercially grown as cash crop in Himachal Pradesh, India. However, its overall production is severely hampered by diamondback moth (Plutella xylostella), a notorious pest. To avoid yield losses caused by this pest, cryIAa gene was introduced into cabbage cv. Pride of India using Agrobacterium-mediated transformation method. In an attempt to maximize the transformation frequency, critical determinant factors such as explant type, pre-incubation and co-cultivation period, and acetosyringone effect were successfully optimized. The highest transformation frequency (4.67% and 14.50%) in cotyledon and hypocotyl explant was achieved with a pre-incubation period of 72h and co-cultivation period of 48h. Furthermore, transformation frequency was enhanced in cotyledon (18.66%) and hypocotyl (32.00%) explants, when selective regeneration medium was fortified with 100µM acetosyringone, respectively. The transgene (cryIAa) integration and copy number were confirmed using PCR and Southern blotting. Reverse transcriptase PCR and quantitative real-time PCR analyses were performed that proved transcriptional expression of cryIAa gene in PCR-positive transgenic events. Transgenic cabbage-fed diamondback moth larvae showed significantly higher mortality, thereby proving transgene effectiveness against insect pest control.

Results from Melanoma Antigen Redirected Vaccine Stimulated Autologous Lymphocytes (MARVSmALo): A pilot study.

e15026 Background: Despite improvements in treatment with targeted agents and immunotherapeutics metastatic melanoma still has a guarded prognosis. Many melanoma cells upregulate the disialoganglioside GD2. Early GD2 chimeric antigen receptor (CAR) T-cell studies in neuroblastoma demonstrated no toxicity but limited ability to expand in vivo. Strategies to expand these GD2.CAR T cells might improve efficacy while retaining safety. This pilot study aimed to evaluate the safety and efficacy of first generation 14g2a.zeta chimeric antigen receptor ( GD2.CAR ) transduced, activated T cells enriched for vaccine specific cytotoxic T-Lymphocytes (tvs-CTL). Methods: Patients with metastatic melanoma in which standard therapy had failed were eligible if they had recovered from effects of prior therapy, did not have rapidly progressive disease, were free of melanoma involving the CNS and did not have a contraindication to receiving Hepatitis B, Polio or DTAP vaccine. Patients received each of these vaccines prior to cell harvest, 4 days before and 28 days after autologous T cell infusion. Patient 1 was treated with 2 x 108 cell dose and patients 2 and 3 were treated with 4x108 cell dose. We used QPCR to measure transgene copy number in patients before and after infusion. Interferon-gamma enzyme linked immunospot (ELISPOT) assay was used to measure the frequencies of tetanus, pertussis, diphtheria, poliovirus and tumor antigens-specific T cells in peripheral blood. Results: GD2.CAR-tvs-CTL were manufactured and infused in 3 patients. Overall the infusions were safe. Seven low grade adverse events possibly related to study participation were reported. The first 2 patients did not demonstrate robust in vivo expansion of GD2.CAR-tvs-CTLs by QPCR and had rapid disease progression. In patient 3 a significant expansion of GD2.CAR-tvs-CTLs, i.e. 18,250 copies/ug genomic DNA was observed on day 7 and cells persisted at 159 copies/ug DNA for up to 12 months (latest measured time point). High pertussis-specific responses were also observed by INF-gamma ELISPOT in this patient starting from day 14 after the vaccination through month 12. Conclusions: We have demonstrated that GD2.CAR T cells expanded and persisted in melanoma patient for up to 12 months. The use of vaccination before blood procurement for T cell manufacture and boosting virus-specific T cell after CAR T cell infusion is a safe strategy and may have helped induced higher transgene levels in one of three patients. Clinical trial information: NCT02482532 .

Effects of viral promoters, the Woodchuck hepatitis post-transcriptional regulatory element, and weakened antibiotic resistance markers on transgene expression in Chinese hamster ovary cells

The product productivity of Chinese hamster ovary (CHO) cells need to be further increased and solved. Promoter and selection marker are essential elements for transgene expression. We investigated the effects of promoters, Woodchuck hepatitis post-transcriptional regulatory (WPRE) element, and selection marker on transgene expression in CHO cells. A weakened form of resistance gene neomycin phosphotransferase (NPT) and WPRE element were cloned into the expression vectors driven by the cytomegalovirus (CMV) and simian virus 40 (SV40) promoters, following by transfecting into CHO cells and screened under G418 pressure. qRT-PCR and Flow cytometry were used to detect the expression level of eGFP gene, and transgene copy number was determined using qPCR technology. Western blot and ELISA were utilized to analyze the expression levels of cetuximab. The weakened NPT gene enhanced eGFP expression both under the CMV or SV40 promoters. Moreover, WPRE, combined with the NPT gene considerably enhanced eGFP expression in CHO cells. Furthermore, the CMV promoter and WPRE combination resulted in higher cetuximab expression levels than CMV promoter alone. However, WPRE could not enhance the transgene expression under the circumstance of CMV promoter and weakened NPT gene combination, suggesting that the effects of the WPRE combined with selection markers were specificity.

Detection and Quantification of Chimeric Antigen Receptor Transgene Copy Number by Droplet Digital PCR versus Real-Time PCR

Chimeric antigen receptor (CAR) T-cell immunotherapy is a new strategy for the treatment of refractory B-cell malignancies; therefore, the rapid and accurate quantification of CAR transgene copy number is essential. Real-time PCR was used for quantifying the copy number of chimeric antigen receptor transgene. Droplet digital PCR (ddPCR) is an absolute quantification method that does not require a standard curve. In this study, key performance parameters of the ddPCR and real-time PCR methods were assessed, including linearity, detection range, the lower limit of detection, repeatability, reproducibility, and accuracy, using a series of gradient diluted standards and clinical peripheral blood samples from CAR T-cell patients. The two platforms showed a good correlation for the standards (Pearson R2=0.9966; P<0.0001) and clinical samples (Pearson R2=0.8952; P<0.0001), and both showed good linearity (R2=0.9996 for ddPCR; R2=0.9984 for real-time PCR) over the detection range. Compared with real-time PCR, ddPCR showed lower intra-assay and interassay CVs for the series of diluted standards, which indicated ddPCR has better repeatability and reproducibility. The limit of detection of ddPCR was lower compared with that of real-time PCR. The combined results suggest that ddPCR is a more promising tool for the detection and quantification of the chimeric antigen receptor transgene copy number.

Small Alphaherpesvirus Latency-Associated Promoters Drive Efficient and Long-Term Transgene Expression in the CNS

Recombinant adeno-associated viruses (rAAVs) are used as gene therapy vectors to treat central nervous system (CNS) diseases. Despite their safety and broad tropism, important issues need to be corrected such as the limited payload capacity and the lack of small gene promoters providing long-term, pan-neuronal transgene expression in the CNS. Commonly used gene promoters are relatively large and can be repressed a few months after CNS transduction, risking the long-term performance of single-dose gene therapy applications. We used a whole-CNS screening approach based on systemic delivery of AAV-PHP.eB, iDisco+ tissue-clearing and light-sheet microscopy to identify three small latency-associated promoters (LAPs) from the herpesvirus pseudorabies virus (PRV). These promoters are LAP1 (404 bp), LAP2 (498 bp), and LAP1_2 (880 bp). They drive chronic transcription of the virus-encoded latency-associated transcript (LAT) during productive and latent phases of PRV infection. We observed stable, pan-neuronal transgene transcription and translation from AAV-LAPs in the CNS for 6 months post AAV transduction. In several CNS areas, the number of cells expressing the transgene was higher for LAP2 than the large conventional EF1α promoter (1,264 bp). Our data suggest that the LAPs are suitable candidates for viral vector-based CNS gene therapies requiring chronic transgene expression after one-time viral-vector administration.

Modeling cross-regulatory influences on monolignol transcripts and proteins under single and combinatorial gene knockdowns in Populus trichocarpa.

Accurate manipulation of metabolites in monolignol biosynthesis is a key step for controlling lignin content, structure, and other wood properties important to the bioenergy and biomaterial industries. A crucial component of this strategy is predicting how single and combinatorial knockdowns of monolignol specific gene transcripts influence the abundance of monolignol proteins, which are the driving mechanisms of monolignol biosynthesis. Computational models have been developed to estimate protein abundances from transcript perturbations of monolignol specific genes. The accuracy of these models, however, is hindered by their inability to capture indirect regulatory influences on other pathway genes. Here, we examine the manifestation of these indirect influences on transgenic transcript and protein abundances, identifying putative indirect regulatory influences that occur when one or more specific monolignol pathway genes are perturbed. We created a computational model using sparse maximum likelihood to estimate the resulting monolignol transcript and protein abundances in transgenic Populus trichocarpa based on targeted knockdowns of specific monolignol genes. Using in-silico simulations of this model and root mean square error, we showed that our model more accurately estimated transcript and protein abundances, in comparison to previous models, when individual and families of monolignol genes were perturbed. We leveraged insight from the inferred network structure obtained from our model to identify potential genes, including PtrHCT, PtrCAD, and Ptr4CL, involved in post-transcriptional and/or post-translational regulation. Our model provides a useful computational tool for exploring the cascaded impact of single and combinatorial modifications of monolignol specific genes on lignin and other wood properties.

Successful Gene Delivery to Cardiac Allograft with Adeno-Associated Viral Vector Using Ex Vivo Storage Perfusion Platform

Cardiac gene therapy remains limited by inefficient transgene delivery with currently available methods (intravenous infusion, transcatheter coronary artery infusion, direct myocardial injection). We previously demonstrated in a porcine heterotopic heart transplant model that cardiac allograft gene transfer can be achieved using ex vivo perfusion with adenovirus (Ad). Adeno-associated viral (AAV) vectors generally achieve lower transgene expression compared to Ad. However, AAV vectors also achieve a significantly longer duration of expression, and thus are more suitable for therapies with genes that are effective even at lower levels but require prolonged expression. The purpose of this study is to evaluate whether ex vivo perfusion can also be used as a platform for cardiac allograft gene therapy using AAV. Heart transplantation was performed using blood type-matched and swine leukocyte antigen-matched Yucatan mini pigs (n=2 transplants). Donor hearts received ex vivo perfusion for two hours with a perfusate including AAV vector bearing the firefly luciferase gene (dosed at 2 × 10^13 and 1 × 10^14 genome-containing vectors for transplants 1 and 2, respectively). The allograft was implanted heterotopically into the recipient abdomen. Recipients were survived for 30 days on oral immunosuppression and then euthanized. Transgene expression in allograft and native recipient tissues was assessed using a luciferase enzymatic assay, and transgene detection was performed using qPCR on DNA extracted using a modified Hirt protocol. Luciferase activity was detected in the allograft in both experiments. While luciferase activity was undetectable in the right and left atria and coronary arteries from the allograft exposed to the lower dose, the higher dose of AAV resulted in detectable luciferase activity throughout the entire heart and at high levels. Luciferase activity was undetectable in all recipient tissues (heart, lung, liver, spleen, and psoas muscle). Additionally, luciferase DNA was detectable on qPCR in allograft specimens (1.5-1.9 × 10^3 transgene copies per 100 ng of DNA). Ex vivo perfusion can be used as a platform for cardiac allograft gene therapy using AAV vectors, with robust transgene expression detectable 30 days following transplant.

The Integration of Multiple Nuclear-Encoded Transgenes in the Green Alga Chlamydomonas reinhardtii Results in Higher Transcription Levels

The integration of genes into the nuclear genome of Chlamydomonas reinhardtii is mediated by Non-Homologous-End-Joining, thus resulting in unpredicted insertion locations. This phenomenon defines ‘the position-effect’, which is used to explain the variation of expression levels between different clones transformed with the same DNA fragment. Likewise, nuclear transgenes often undergo epigenetic silencing that reduces their expression; hence, nuclear transformations require high-throughput screening methods to isolate clones that express the foreign gene at a desirable level. Here, we show that the number of integration sites of heterologous genes results in higher mRNA levels. By transforming both a synthetic ferredoxin-hydrogenase fusion enzyme and a Gaussia-Luciferase reporter protein, we were able to obtain 33 positive clones that exhibit a wide range of synthetic expression. We then performed a droplet-digital polymerase-chain-reaction for these lines to measure their transgene DNA copy-number and mRNA levels. Surprisingly, most clones contain two integration sites of the synthetic gene (45.5%), whilst 33.3% contain one, 18.1% include three and 3.1% encompass four. Remarkably, we observed a positive correlation between the raw DNA copy-number values to the mRNA levels, suggesting a general effect of which transcription of transgenes is partially modulated by their number of copies in the genome. However, our data indicate that only clones harboring at least three copies of the target amplicon show a significant increment in mRNA levels of the reporter transgene. Lastly, we measured protein activity for each of the reporter genes to elucidate the effect of copy-number variation on heterologous expression.

Hipscs Derived Cardiomyocytes Overexpressing Deoxy ATP to Restore Cardiac Function

Reduced contractility, caused either by dysfunction or loss of cardiomyocytes, is the leading cause of systolic heart failure (HF). We have demonstrated that deoxy-ATP (dATP) improves systolic cardiac contractility by activating myosin without affecting the diastolic function, suggesting a promising candidate to mitigate cardiac dysfunction. Endogenously, Ribonucleotide Reductase (RNR), a rate-limiting enzyme for de novo synthesis of deoxynucleotides, produces dATP. The goal is to overexpress RNR in human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) to create cells with elevated dATP levels for use as a novel therapeutic strategy to improve contractility and regeneration in hearts following myocardial infarct. We used CRISPR-Cas9 gene editing to integrate an RNR expression cassette at the AAVS1 locus in hiPSCs WTC11 cell line (#GM25256, Coriell Institute). A double mutant form (RNR-DM) was generated by site-directed mutagenesis to limit proteolytic degradation of RNR in post-mitotic CMs. RNR subunits—R1 and R2-DM—intervened by a self-cleavage peptide P2A were expressed under a strong constitutively expressed CAG promoter. The integration of RNR cassette at the AAVS1 locus in hiRNR clones were confirmed by PCR genotyping, sequencing, transgene transcript, and protein expression. The stability and function of RNR-DM transgene were assessed in hiRNR-CMs, differentiated from the hiRNR clone. hiRNR-CMs expressed elevated levels of R1 and R2 subunits as compared to control WTC11-CMs (N=3, where N is an independent run of cardiac differentiation) by immunoblotting. This resulted in an elevation of dATP/ATP ratio (3.4±1.9 fold) (N=2) for hiRNR-CMs vs. WTC11-CMs, as detected by Mass Spectrometry. Cardiomyocyte shortening measurements by video-based IonOptix were increased (2.03±0.7 fold) in hiRNR CMs as compared to WTC11-CMs (N=2). Currently, we are characterizing the hiRNR-CMs, including DNA stability, apoptosis, cell division, calcium cycling, hypertrophy, and structural maturation for future studies of transplantation into models of HF.

Variation in Spontaneous Activity and Visual Evoked Response in Primary Visual Cortex of the S334ter-3 Rats

S334ter-3 retinal degeneration (RD) rats have been widely used to investigate degenerative diseases of the retina. In this model, morphological and electrophysiological changes have been observed in the retina, superior colliculus and primary visual cortex (V1). In this study, experimental rats (S334ter-3) carried one copy of the mutant transgene. We measured the extracellular responses in the primary visual cortex to three stimulus contrast levels (spontaneous activity, medium contrast, and high contrast) at the preferred parameters of each recorded cell under classical receptive field (CRF) stimulation. Then we compared the responses (spontaneous activity and the visual evoked responses) in RD rats with those in wildtype rats. Our results show that V1 cells in the RD group exhibit stronger spontaneous activity but weaker stimulus-evoked responses at medium and high contrasts. At the same time, compared with WT group, RD group also showed a narrow dynamic range. These results indicate the decrease in discriminating the stimuli contrast and loss in responses and lower signal to noise ratio after retina degeneration.

Predicting favorable landing pads for targeted integrations in Chinese hamster ovary cell lines by learning stability characteristics from random transgene integrations

Chinese Hamster Ovary (CHO) cell lines are considered to be the preferred platform for the production of biotherapeutics, but issues related to expression instability remain unresolved. In this study, we investigated potential causes for an unstable phenotype by comparing cell lines that express stably to such that undergo loss in titer across 10 passages. Factors related to transgene integrity and copy number as well as the genomic profile around the integration sites were analyzed. Horizon Discovery CHO-K1 (HD-BIOP3) derived production cell lines selected for phenotypes with low, medium or high copy number, each with stable and unstable transgene expression, were sequenced to capture changes at genomic and transcriptomic levels. The exact sites of the random integration events in each cell line were also identified, followed by profiling of the genomic, transcriptomic and epigenetic patterns around them. Based on the information deduced from these random integration events, genomic loci that potentially favor reliable and stable transgene expression were reported for use as targeted transgene integration sites. By comparing stable vs unstable phenotypes across these parameters, we could establish that expression stability may be controlled at three levels: 1) Good choice of integration site, 2) Ensuring integrity of transgene and observing concatemerization pattern after integration, and 3) Checking for potential stress related cellular processes. Genome wide favorable and unfavorable genomic loci for targeted transgene integration can be browsed at https://www.borthlabchoresources.boku.ac.at/