Fluorescence-assisted sequential insertion of transgenes (FASIT): an approach for increasing specific productivity in mammalian cells (original) (raw)
Related papers
Molecular Biotechnology, 2017
Production of recombinant pharmaceutical proteins has made a great contribution to modern biotechnology. At present, quick advances in protein expression lead to the enhancement of product quantity and quality as well as reduction in timescale processing. In the current study, we assessed the expression level of recombinant human follicle-stimulating hormone (rhFSH) in adherent and suspension Chinese hamster ovary (CHO) cell lines by cultivation in serum-containing and chemically defined, protein-free media. The expression cassette entailing FSH subunits was transfected to CHO/dhfr-and CHO DG44 cell lines, and gene amplification was achieved using dihydrofolate reductase (DHFR)/methotrexate (MTX) system. Afterward, the expression level of rhFSH was studied using real-time PCR, Western blotting and ELISA. Our achievements revealed that stepwise increase in MTX [up to 2000 nano-molar (nM)] leads to boost the expression level of rhFSH mRNA in both cell lines, although DG44 have better results, as mRNA expression level reached 124.8-and 168.3-fold in alpha and beta subunits, respectively. DG44 cells have also the best protein production in 2000 nM MTX, which reached 1.7-fold in comparison with that of the mock group. According to the above results and many advantages of protein-free media, DG44 is preferable cell line for future steps. Keywords CHO/dhfr-Á DG44 Á Follicle-stimulating hormone (FSH) Á Gene amplification systems Á Protein-free media Á Recombinant protein expression Á Serumcontaining media Electronic supplementary material The online version of this article (
Theriogenology, 2012
The objective was to evaluate the effects of cell cycle inhibitors (6-dimethylaminopurine [DMAP], and dehydroleukodine [DhL]) on transgene expression efficiency and on mosaic expression patterns of IVF bovine zygotes cytoplasmically injected with oolema vesicles coincubated with transgene. The DNA damage induced by the transgene or cell cycle inhibitors was measured by detection of phosphorylated histone H2AX foci presence (marker of DNA double-stranded breaks). Cloning of egfp blastomeres was included to determine continuity of expression after additional rounds of cellular division. The pCX-EGFP [enhanced green fluorescent protein gene (EGFP) under the chimeric cytomegalovirus IE-chicken--actin enhancer promoter control] gene plasmid (50 ng/L) was injected alone (linear or circular exogenous DNA, leDNA and ceDNA, respectively) or associated with ooplasmic vesicles (leDNA-v or ceDNA-v). The effects of 2 mM DMAP or 1 M DhL for 6 h (from 15 to 21 h post IVF) was evaluated for groups injected with vesicles. The DMAP increased (P Ͻ 0.05) egfp homogenous expression relative to transgene alone (21%, 18%, and 11% for leDNA-v ϩ DMAP, leDNA-v, and leDNA, respectively) and also increased (P Ͻ 0.05) the phosphorylated histone H2AX foci area. Expression of egfp was higher (P Ͻ 0.05) for linear than for circular pCX-EGFP, and egfp blastocyst rates were higher (P Ͻ 0.05) for groups injected with linear transgene coincubated with vesicles than for linear transgene alone (95%, 77%, 84%, and 52% for leDNA-v ϩ DMAP, leDNA-v ϩ DhL, leDNA-v, and leDNA, respectively). Moreover, DMAP tended to improve egfp blastocysts rates for both circular and linear transgenes. Based on fluorescent in situ hybridization (FISH) analysis, there was evidence of integration in egfp embryos. Finally, clones derived from leDNA-v ϩ DMAP had the highest egfp expression rates (96%, 65%, and 65% for leDNA-v ϩ DMAP, leDNA-v, and leDNA, respectively). Transgenesis by cytoplasmic injection of leDNA-v ϩ DMAP is a promising alternative for transgenic animal production.
Human Reproduction Update, 1998
Genes encoding the common gonadotrophin α subunit and follicle stimulating hormone (FSH)-specific β subunit were isolated from a DNA library derived from human fetal liver cells, and inserted into separate expression vectors containing a selectable/amplifiable gene. These vectors were inserted into the genome of the Chinese hamster ovary cell line, resulting in expression of large amounts of biologically active human (h)FSH. This cell line was cultured on microcarrier beads in a large-scale bioreactor. hFSH in the cell culture supernatant was purified to homogeneity by a multistep process. The mature β subunit had seven fewer amino acid residues than reported in the literature and three other differences were found in the sequence. Similar oligosaccharide structures were present on recombinant (r)-hFSH and a purified urinary (u)-hFSH preparation. In-vitro and in-vivo, the biological activities of u-and r-hFSH were indistinguishable. r-hFSH was formulated in ampoules containing 75 IU FSH activity (∼7.5 µg FSH), which accounts for >99% of the protein content of the preparation. Studies in nonhuman primates and human volunteers showed the pharmacokinetics of u-and r-hFSH to be similar. In healthy volunteers, r-hFSH stimulated follicular development and induced significant increases in serum oestradiol and inhibin. Clinical experience with r-hFSH has shown it is more effective at stimulating ovarian follicle growth than urinary gonadotrophins. It is also effective at initiating spermatogenesis when given together with human chorionic gonadotrophin.
A system for site-specific integration of transgenes in mammalian cells
PLOS ONE
Mammalian cell expression systems are the most commonly used platforms for producing biotherapeutic proteins. However, development of recombinant mammalian cell lines is often hindered by the unstable and variable transgene expression associated with random integration. We have developed an efficient strategy for site-specific integration of genes of interest (GOIs). This method enables rapid and precise insertion of a gene expression cassette at defined loci in mammalian cells, resulting in homogeneous transgene expression. We identified the Hipp11 (H11) gene as a "safe harbor" locus for gene knock-in in CHO-S cells. Using clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 mediated homologous recombination, we knocked in a DNA cassette (the landing pad) that includes a pair of PhiC31 bacteriophage attP sites and genes facilitating integrase-based GOI integration. A master cell line, with the landing pad inserted correctly in the H11 locus, was established. This master cell line was used for site-specific, irreversible recombination, catalyzed by PhiC31 integrase. Using this system, an integration efficiency of 97.7% was achieved with green fluorescent protein (GFP) after selection. The system was then further validated in HEK293T cells, using an analogous protocol to insert the GFP gene at the ROSA26 locus, resulting in 90.7% GFP-positive cells after selection. In comparison, random insertion yielded 0.68% and 1.32% GFP-positive cells in the CHO-S and HEK293T cells, respectively. Taken together, these findings demonstrated an accurate and effective protocol for generating recombinant cell lines to provide consistent protein production. Its likely broad applicability was illustrated here in two cell lines, CHO-S and HEK293T, using two different genomic loci as integration sites. Thus, the system is potentially valuable for biomanufacturing therapeutic proteins.
Molecular Human Reproduction, 1996
o whom correspondence should be addressed Follicle stimulating hormone (FSH) is a heterodimeric glycoprotein hormone produced in the anterior pituitary gland. The hormone is essential in the regulation of reproductive processes, such as follicular development and ovulation. It is clinically used for treatment of anovulation and in assisted reproduction technologies such as in-vrtro fertilization (IVF) and intracytoplasmic sperm injection (ICSI). Until recently, the only source for human FSH has been the urine from post-menopausal women. Such a natural source implies limited availability and potential product variability. Thus, we have cloned the genes encoding the a-and {J-subunrts of human FSH and transfected these into Chinese hamster ovary (CHO) cells. A CHO-clone was isolated capable of secreting intact glycosylated FSH with identical amino acid sequences to natural FSH. This cell line was grown in perfusion culture and enabled us to isolate highly pure FSH (>99%). The complexity of the charge distribution of human recombinant FSH was demonstrated by isoelectric focusing. The observed microheterogeneity is caused by the large number of carbohydrate chain structures which are added to the four potential glycosylation sites in the afJ-dimer. Furthermore, the carbohydrates show a variation in their degree of sialylation which reflects the different pi values of the individual isohormones. Despite the complexity of post-translational modification, the isoform distribution of recombinant FSH produced in a CHO-cell line and grown in perfusion culture is surprisingly similar to that observed with pituitary FSH and urinary FSH. In conclusion, we have shown that FSH-gene transfected CHO-cells are capable of stable serumfree production of recombinant FSH. A process has been developed which assures the consistent and reproducible production of highly-purified recombinant FSH.
Expression of Recombinant Human Follicle-Stimulating Hormone in the Mammary Gland of Transgenic Mice
Molecular Biotechnology, 2006
In this article, we describe the use of a caprine β-lactoglobulin (βLG) expression cassette previously obtained by our group to target the expression of the human follicle-stimulating hormone (hFSH) to the mammary gland of transgenic mice. The hFSH is a pituitary glycoprotein composed of two subunits (α and β). At present, this protein is obtained from mammalian cellular fermentors, and it is extensively used in the treatment of human infertility. Four lines of double (hFSHα/β) transgenic mice that stably transmitted the transgenes were obtained, and hFSHα and hFSHβ mRNA was detected by reverse transcriptase-polymerase chain reaction in the mammary gland of lactating females from all four transgenic lines. The hFSH protein was present in the mammary gland of the lactating females, but could not be detected in the milk by Western blot, probably as a result of low levels of transgene expression.
Transient Recombinant Protein Expression in Mammalian Cells: the Role of mRNA Level and Stability
2009
Transient gene expression (TGE) is a rapid method for generating recombinant proteins in mammalian cells, but the volumetric productivities for secreted proteins in transiently transfected CHO DG44 cells are typically more than an order of magnitude lower than the yields achieved with recombinant CHO-derived cell lines. The goals of the thesis are to identify the limitations to higher TGE yields in CHO DG44 cells and to find possible solutions to overcome the problems. Initially an attempt was made to enhance TGE production by increasing the amount of transfected plasmid DNA. However, this approach did not result in increased recombinant protein levels; on the contrary, transfection with an excess of plasmid DNA (> 1.25 μg/ml) had a negative impact on transgene mRNA levels and protein production. Moreover, it was also observed that recombinant protein yield was strongly dependent on the mRNA level. Therefore, three strategies aimed at increasing the amount of transgene mRNA were investigated. For the first approach, transfected cells were exposed to hypothermic conditions during the production phase. It was already known that lower temperatures increase protein production several fold in recombinant CHO DG44-derived cell lines. The second strategy involved the treatment of transfected cells with valproic acid, a histone deacetylase inhibitor that reduces the effects of gene silencing. The third approach aimed to increase transgene mRNA levels by overexpressing transcription factors and growth factors. With the first two strategies recombinant antibody yields of 60-80 mg/L were achieved whereas the untreated control transfections produced only 5-10 mg/L. Combination of the two strategies led to the production of 90 mg/L of antibody. Moreover, in the treated cultures, the steady-state level of transgene mRNA was 3-5 times higher than in the untreated cultures and remained stable up to 6 days post-transfection. Using the third approach, the increase in recombinant protein production was moderate and transgene mRNA amounts were only 2-fold higher in treated samples compared to the control. When specific proteins such as c-fos, c-jun, NF-kB, and acidic fibroblast growth factor (aFGF) were overexpressed the recombinant antibody production was 20 mg/L compared to 5 mg/L for the control transfection. Overexpression of either a transcription factor or a growth factor in combination with treatment with valproic acid allowed the recombinant protein yield to reach 90 mg/L. However, the benefit of the overexpressed factors was minimal compared to the effect of valproic acid alone. In conclusion, it was demonstrated that the level and stability of transgene mRNA are important factors for increasing volumetric yields in transiently transfected CHO DG44 cells. Furthermore, three approaches aimed to increase mRNA amounts were tested. Exposure to hypothermic conditions and treatment with valproic acid were the two best strategies tested. Both are simple, cost-effective, and scalable making transient gene expression in CHO DG44 cells a feasible alternative for rapid production of gram amounts of recombinant protein.
A GFP-based screen for growth-arrested, recombinant protein-producing cells
Biotechnology and Bioengineering, 2002
The growth of anchorage-dependent Chinese hamster ovary (CHO) cells is arrested upon serum deprivation; however, a portion of these cells remain viable for extended time periods in serum-free culture. This work presents a strategy to both rapidly generate a heterogeneous population of CHO cells as well as to select for subpopulations that remain robust and continue to produce recombinant protein when their growth is arrested. Stable expression of recombinant proteins in mammalian cells is often a tedious and time-consuming process because only a small percentage of transfected cells will express suf®cient quantities of protein. To overcome the limitations associated with standard transformation and selection methods, bicistronic retroviral expression technology was used. First, bicistronic retroviral constructs encoding for both interferon gamma (IFN-c), the model therapeutic protein, and green uorescent protein (GFP), the quantitative selectable marker, were generated. Next, recombinant retroviruses were obtained from transient transfection of a helper-cell line and were used to infect susceptible CHO cells. Cells with the bicistronic expression module stably integrated into their genome¯uoresce green and could thereby be easily isolated by¯uorescence-activated cell sorting. Upon subjecting successfully infected cells to serum withdrawal, signi®cant declines in cell viability and GFP expression occurred. After imposing this selection pressure on the cells for 8 days, GFP producers were isolated from the survivors by¯uorescence-activated cell sorting and expanded. To evaluate the effectiveness of the screening process, the selected cells were exposed to a second round of serum deprivation. Unlike the original cell population from which it was derived, the subpopulation remained robust and continued to stably express both GFP and IFN-c throughout the extended period of serum-free culture. Within 2 weeks, cells selected for recombinant protein production under serum-free conditions were successfully generated and isolated. ã