6 research outputs found
CHO genome mining for synthetic promoter design
Synthetic promoters are an attractive alternative for use in mammalian hosts such as CHO cells as they can be designed de novo with user-defined functionalities. In this study, we describe and validate a method for bioprocess-directed design of synthetic promoters utilizing CHO genomic sequence information. We designed promoters with two objective features, (i) constitutive high-level recombinant gene transcription, and (ii) upregulated transcription under mild hypothermia or late-stage culture. CHO genes varying in transcriptional activity were selected based on a comparative analysis of RNA-Seq transcript levels in normal and biphasic cultures in combination with estimates of mRNA half-life from published genome scale datasets. Discrete transcription factor regulatory elements (TFREs) upstream of these genes were informatically identified and functionally screened in vitro to identify a subset of TFREs with the potential to support high activity recombinant gene transcription during biphasic cell culture processes. Two libraries of heterotypic synthetic promoters with varying TFRE combinations were then designed in silico that exhibited a maximal 2.5-fold increase in transcriptional strength over the CMV-IE promoter after transient transfection into host CHO-K1 cells. A subset of synthetic promoters was then used to create stable transfectant pools using CHO-K1 cells under glutamine synthetase selection. Whilst not achieving the maximal 2.5-fold increase in productivity over stable pools harboring the CMV promoter, all stably transfected cells utilizing synthetic promoters exhibited increased reporter production - up to 1.6-fold that of cells employing CMV, both in the presence or absence of intron A immediately downstream of the promoter. The increased productivity of stably transfected cells harboring synthetic promoters was maintained during fed-batch culture, with or without a transition to mild hypothermia at the onset of stationary phase. Our data exemplify that it is important to consider both host cell and intended bioprocess contexts as design criteria in the de novo construction of synthetic genetic parts for mammalian cell engineering
Design of synthetic promoters for controlled expression of therapeutic genes in retinal pigment epithelial cells
Ageârelated macular degeneration (AMD) associated with dysfunction of retinal pigment epithelial (RPE) cells is the most common cause of untreatable blindness. To advance gene therapy as a viable treatment for AMD there is a need for technologies that enable controlled, RPEâspecific expression of therapeutic genes. Here we describe design, construction and testing of compact synthetic promoters with a preâdefined transcriptional activity and RPE cell specificity. Initial comparative informatic analyses of RPE and photoreceptor (PR) cell transcriptomic data identified conserved and overrepresented transcription factor regulatory elements (TFREs, 8â19âbp) specifically associated with transcriptionally active RPE genes. Both RPEâspecific TFREs and those derived from the generically active cytomegalovirusâimmediate early (CMVâIE) promoter were then screened in vitro to identify sequence elements able to control recombinant gene transcription in model induced pluripotent stem (iPS)âderived and primary human RPE cells. Two libraries of heterotypic synthetic promoters varying in predicted RPE specificity and transcriptional activity were designed de novo using combinations of up to 20 discrete TFREs in series (323â602âbp) and their transcriptional activity in model RPE cells was compared to that of the endogenous BEST1 promoter (661âbp, plus an engineered derivative) and the highly active generic CMVâIE promoter (650âbp). Synthetic promoters with a highpredicted specificity, comprised predominantly of endogenous TFREs exhibited a range of activities up to 8âfold that of the RPEâspecific BEST1 gene promoter. Moreover, albeit at a lower predicted specificity, synthetic promoter transcriptional activity in model RPE cells was enhanced beyond that of the CMVâIE promoter when viral elements were utilized in combination with endogenous RPEâspecific TFREs, with a reduction in promoter size of 15%. Taken together, while our data reveal an inverse relationship between synthetic promoter activity and cellâtype specificity, cell contextâspecific control of recombinant gene transcriptional activity may be achievable
Increased recombinant adenoâassociated virus production by HEK293 cells using small molecule chemical additives
Recombinant adeno-associated virus (rAAV) has established itself as a highly efficacious gene delivery vector with a well characterised safety profile allowing broad clinical application. Recent successes in rAAV-mediated gene therapy clinical trials will continue to drive demand for improved rAAV production processes to reduce costs. Here, we demonstrate that small molecule bioactive chemical additives can significantly increase recombinant AAV vector production by human embryonic kidney (HEK) cells up to three-fold. Nocodazole (an anti-mitotic agent) and M344 (a selective histone deacetylase inhibitor) were identified as positive regulators of rAAV8 genome titre in a microplate screening assay. Addition of nocodazole to triple-transfected HEK293 suspension cells producing rAAV arrested cells in G2/M phase, increased average cell volume and reduced viable cell density relative to untreated rAAV producing cells at harvest. Final crude genome vector titre from nocodazole treated cultures was >2-fold higher compared to non-treated cultures. Further investigation showed nocodazole addition to cultures to be time critical. Genome titre improvement was found to be scalable and serotype independent across two distinct rAAV serotypes, rAAV8 and rAAV9. Furthermore, a combination of M344 and nocodazole produced a positive additive effect on rAAV8 genome titre, resulting in a three-fold increase in genome titre compared to untreated cells
Production of trimeric SARSâCoVâ2 spike protein by CHO cells for serological COVIDâ19 testing
We describe scalable and costâefficient production of full length, Hisâtagged severe acute respiratory syndrome coronavirus 2 (SARSâCoVâ2) spike glycoprotein trimer by Chinese hamster ovary (CHO) cells that can be used to detect SARSâCoVâ2 antibodies in patient sera at high specificity and sensitivity. Transient production of spike in both human embryonic kidney (HEK) and CHO cells mediated by polyethyleneimine was increased significantly (up to 10.9âfold) by a reduction in culture temperature to 32°C to permit extended duration cultures. Based on these data GSâCHO pools stably producing spike trimer under the control of a strong synthetic promoter were cultured in hypothermic conditions with combinations of bioactive small molecules to increase yield of purified spike product 4.9âfold to 53âmg/L. Purification of recombinant spike by Niâchelate affinity chromatography initially yielded a variety of coâeluting protein impurities identified as host cell derived by mass spectrometry, which were separated from spike trimer using a modified imidazole gradient elution. Purified CHO spike trimer antigen was used in enzymeâlinked immunosorbent assay format to detect immunoglobulin G antibodies against SARSâCoVâ2 in sera from patient cohorts previously tested for viral infection by polymerase chain reaction, including those who had displayed coronavirus disease 2019 (COVIDâ19) symptoms. The antibody assay, validated to ISO 15189 Medical Laboratories standards, exhibited a specificity of 100% and sensitivity of 92.3%. Our data show that CHO cells are a suitable host for the production of larger quantities of recombinant SARSâCoVâ2 trimer which can be used as antigen for mass serological testing
KARAKTERISTIK DAN KUALITAS SEMEN BERBAGAI GALUR AYAM KEDU (Characteristic and Cemen Quality at Various Lines of Kedu Chicken)
ABSTRAK
Ayam kedu adalah ayam lokal yang mempunyai ciriâciri khas dan telah lama terdapat di
Desa Kedu, Kecamatan KeduâKabupaten Temanggung. Tujuan penelitian adalah mengamati
derajat keasaman (pH), daya hidup dan mortalitas semen ayam Kedu hitam daging hitam (HH),
Kedu putih daging putih (PP) dan Kedu hitam daging putih (HP). Serta untuk mengetahui
karakteristik semen dari berbagai galur warna ayam Kedu.Materi yang digunakan dalam
penelitian ini adalah semen yang diambil dari 15 ekor ayam kedu jantan, terdiri dari 5 ekor ayam
kedu hitam daging hitam (HH), 5 ekor ayam kedu hitam daging putih (HP) dan 5 ayam kedu
putih daging putih (PP), dengan kisaran umur 8-12 bulan. Hasil penelitian menunjukkan bahwa
rata-rata abnormalitas sperma ayam kedu HH, HP dan PP adalah : 12,87%, 11,84% dan 11,82%
tidak berbeda nyata. Karakteristik semen dari berbagai galur warna ayam kedu yang meliputi
volume, konsentrasi dan abnormalitas tidak berbeda nyata. Hasil uji statistik terhadap derajat
keasaman (pH), daya hidup dan persentase mortalitas semen berdasarkan warna bulu ayam
Kedu tidak menunjukkan perbedaan (Pâ„0,05). Hasil penelitian dapat disimpulkan bahwa derajat
keasaman (pH), daya hidup semen dan mortalitas semen ayam Kedu berdasarkan warna bulu
tidak berbeda.
Kata kunci : Ayam Kedu, Warna bulu, kualitas semen, Karakteristik Semen.
ABSTRACT
Kedu chicken is local chicken which have typical characteristic and there was in
countryside of Kedu, district of Kedu-Temanggung regency. The aim of research was to
perceive degree of acidity (pH), energy live and cemen mortality of black Kedu chicken of black
flesh (HH), White Kedu of white meat (PP) and of Kedu black of white meat (HP). And also to
know cemen characteristic from various chicken colour galur of Kedu. Items which used in this
research was taken away from by cemen of 15 kedu chicken male, consist of 5 Kedu chicken of
kedu black of black flesh (HH), 5 kedu chicken black of white meat (HP) and 5 Kedu chicken
white of white meat (PP), with of old age was 8-12 months. The result of research indicated that
mean of abnormality Kedu chicken sperm of HH, HP and of PP was : 12,87%, 11,84% and
11,82% non differ significant. The cemen characteristic from various chicken colour lines of
kedu covering volume, and concentration of abnormality was not differ significant. Result of
statistical test to acidity degree (pH), energy live and percentage of cemen mortality pursuant to
quill colour of Kedu was not show difference (Pâ„0,05). Result of research can be concluded that
acidity degree (pH), life energy cemen and cemen mortality of Kedu chicken pursuant to fur
colour was not differ.
Keywords : Kedu chicken, Colour Fur, cemen quality, Characteristic of cemen
Engineering of the CMV promoter for controlled expression of recombinant genes in HEK293 cells
Expression of recombinant genes in HEK293 cells is frequently utilized for production of recombinant proteins and viral vectors. These systems frequently employ the cytomegalovirus (CMV) promoter to drive recombinant gene transcription. However, the mechanistic basis of CMV-mediated transcriptional activation in HEK293 cells is unknown and consequently there are no strategies to engineer CMV for controlled expression of recombinant genes. Extensive bioinformatic analyses of transcription factor regulatory elements (TFREs) within the human CMV sequence and transcription factor mRNAs within the HEK293 transcriptome revealed 80 possible regulatory interactions. Through in vitro functional testing using reporter constructs harboring discrete TFREs or CMV deletion variants we identified key TFRE components and clusters of TFREs (cis-regulatory modules) within the CMV sequence. Our data reveal that CMV activity in HEK293 cells is a function of the promoters various constituent TFREs including AhR:ARNT, CREB, E4F, Sp1, ZBED1, JunB, c-Rel, and NF-ÎșB. We also identified critical Sp1-dependent upstream activator elements near the transcriptional start site that were required for efficient transcription and YY1 and RBP-JÎș binding sites that mediate transrepression. Our study shows for the first time that novel, compact CMV-derived promoters can be engineered that exhibit up to 50% higher transcriptional efficiency (activity per unit DNA sequence) or 14% increase in total activity compared to the wild-type counterpart