Mammalian cells

Mammalian cells are cells of higher eukaryotes. Eukaryotic cells have the ability to modify proteins post-translationally, and they contain a large number of membrane bound compartments such as mitochondria, endoplasmatic reticulum, and the Golgi apparatus. Compared to microbes, mammalian cells are fragile, have a slow doubling time (app. 24h), and need complex and expensive growth media. The cells are also easily contaminated with mycoplasma, therefore it is necessary to work as sterile as possible, when handling mammalian cells (1). So what is the deal with these high maintenance cells?

Mammalian cell factories

Mammalian cell cultures represent a suitable and stable gene expression system and are often used as cell factories for production of biopharmaceuticals. Heterologous protein expression in an appropriate host is central in production of biopharmaceuticals. Heterologous proteins require complex post-translational modifications such as glycosylation, gamma-carboxylation, and site specific proteolysis, which only mammalian cells are capable of performing. Moreover, mammalian cells have the unique capability to authentically process, fold and modify secreted human proteins (1). Mammalian cell cultures are therefore widely used for production of therapeutic proteins such as monoclonal antibodies, growth hormones, and cytokines used for a wide array of diseases(4). The effect of post-translational modifications are protein stability, proper ligand binding, and reduced risk of immunogenicity (2), and most of the therapeutic proteins approved and currently in development are therefore modified (3). However, the genetic tools used for constructing mammalian cells vectors are based on outworn methods, and since 60-70% of all recombinant protein pharmaceuticals are produced in mammalian cells, there is a desperate need for simpler and more efficient cloning techniques (5).

The U-2 OS cell line

The U-2 OS cell line, originally known as the 2T line, is an immortalized human-derived cell line that was established in 1964. An immortalized cell line has acquired the ability to proliferate indefinitely through either random mutation or modifications such as artificial expression of the telomerase gene. Several cell lines are well established as representatives of certain cell types. U-2 OS cells show epithelial adherent morphology, and no viruses have been detected in the cell line (17). In comparison, the HeLa cell line contains the well known HPV virus. U-2 OS cells are also very good-looking in the microscope, and therefore this cell line was chosen for the proof of concept of the Plug 'n’ Play assembly standard for mammalian cells.

Transient transfection

Transient expression is the ability to express heterologous DNA during a short period of time, which allows fast production of a desired protein. A high copy number of plasmids are introduced into the cells, and expression may be transitory over a period of time until the DNA is lost from the population. This allows for protein characterization or to verify the integrity, functionality, and the efficiency of different recombinant vectors. Production of a large amount of recombinant protein has been reported for transient expression systems at large scale. A small number of the transfected cells may incorporate the exogenous DNA into their genome by recombination leading to a stable transfection of a gene (6).

Typically mammalian expression vectors have a multiple cloning site (MCS). The gene of interest (GOI) to be inserted into the MCS is therefore required to hold restriction sites compatible with the expression vector. The insertion of the GOI is achieved by digestion and ligation and this classical cloning method can be quite cumbersome. Furthermore, the integration of the gene of interest in the expression vector by restriction enzymes and ligases can have a low efficiency as well as provide a high number of false-positive transformants (6).

Filamentous Fungi

Fungi are a diverse group of organisms, whose biological activities affect our daily life in many ways. The filamentous fungi are in particular of great importance in production of medicine, in the industry, in agriculture, and in basic biological research. Filamentous fungi for example produce a diverse array of secondary metabolites, which are of interest in the pharmaceutical sciences as a prolific source of chemical compounds for the development of new drugs. Some of the filamentous fungal species are pathogenic to humans, whereas others have value in the production of antibiotics such as penicillin. Fungi are therefore of great importance industrially as well as in our daily life.

Growth of filamentous fungi

The vegetative growth of filamentous fungi starts in the right conditions with the germination of a spore. The spore germination leads to formation of hyphae (7). A fungal hyphae is a long tubular modular structure composed of individual cells (8). Hyphae extend only at their tips and are typically divided into individual cellular compartments by the formation of septa as shown in the figure below (9).

Filamentous fungi grow by the polar extension of hyphae and multiply by branching (10). The branched hyphae form a network of interconnected cells called a mycelium (7). The mycelium forms a radially symmetric colony that expands over a large area until growth is limited by for example lack of nutrients (7,11). The fungal mycelium appears to be a formless collection of corresponding vegetative cells. However, the various cells within the mycelium interact to form an ordered network with different hyphae or cells playing distinct roles in the acquirement of nutrients from the environment (7).

Aspergillus nidulans

The filamentous fungus Aspergillus nidulans is a model organism and therefore in contrast to most other aspergilla, has a well characterized sexual cycle and a well-developed genetic tools for manipulation (9,12). Furthermore, in A. nidulans the parasexual cycle has been extensively utilized. Parasexual genetics involves examination of recombination in the absence of sexual reproduction.

The genetic analysis has produced a deep understanding of both the physiology of Aspergilli and the organisation of the genome (13). This research has advanced the study of eukaryotic cellular physiology and contributed to our understanding metabolic regulation, development, DNA repair, morphogenesis, and human genetic diseases (12). Furthermore, the recent sequencing of the complete genome of A. nidulans has created a tremendous potential to obtain insight into important aspects of fungal biology such as transcriptional regulation, secondary metabolite production and pathogenicity (14).

Gene targeting in fungi

Gene targeting facilitates precise genome manipulations, called site directed alterations. They are performed by deletions, replacements and insertions at a target locus. Gene targeting is achieved by transforming fungi with a suitable linear DNA fragment that contains sequences that are identical to the target site in the genome, see figure below. Fungi can integrate linear DNA fragments in its genome by repair of double stranded breaks (15).

Two mechanisms for repair of double stranded breaks ensure that a linear piece of DNA is integrated into the fungal genome; homologous recombination (HR) and non homologous end joining (NHEJ), also called illegitimate recombination (15). HR involves interactions between homologous sequences, whereas NHEJ involves ligation of the ends independently of DNA homology (16). Precise genome manipulation can often be tedious and time-consuming, because fungi appear to favour NHEJ over HR resulting in low gene targeting efficiencies (15).

References

[1] Mueller, P.P., Wirth, D., Unsinger, J. & Hauser, H., 2003. Genetic Approaches to Recombinant Protein Production in Mammalian Cells. In Handbook of Industrial Cell Culture. Humana Press Inc. pp.21-49.

[2] Browne, SM., Al-Rubeai, M. (2007). Selection methods for high-producing mammalian cell lines. TRENDS in Biotechnology, 25, 425-432.

[3] Walsh, W., Jefferis, R. (2006). Post-translational modifications in the context of therapeutic proteins. Nature Biotechnology, 24, 1241-1252.

[4] Xie, L., Zhou, W., & Robinson, D.,2003. Protein production by large-scale mammalian cell culture. S.C. Makrides (Ed.) Gene Transfer and Expression in Mammalian Cells. Elsevier Science B.V.

[5] Wurm, F. M. (2004). Production of recombinant protein therapeutics in cultivated mammalian cells. Nature biotechnology, 22(11), pp. 1393-8.

[6] Bollati-Fogolín, M. & Comini, M.A., 2008. Cloning and expression of heterologous proteins in animal cells. In Animal Cell Technology. Taylor & Francis Group. pp.39-73.

[7] Adams, T.H., Wieser, J.K. & Yu, J.-H., 1998. Asexual Sporulation in Aspergilus nidulans. Microbiology and Molecular Biology Reveiws, vol. 62, no. 1, pp.35-54.

[8] Harris, S.D., 1997. The Duplication Cycle in Asoergillus nidulans. Fungal Genetics and Biology, vol. 22, no. 1, pp.1-12.

[9] Harris, S.D. et al., 2009. Morphology and development in Aspergillus nidulans: A complex puzzle. Fungal Genetics and Biology, vol. 46, no. 1, sup. 1, pp.S82-92.

[10] Timberlake, W.E., 1990. Molecular Genetics of Aspergillus Development. Annual Review of Genetics, vol. 24, pp.5-36.

[11] Nielsen, J.B., 2008. Understanding DNA repair in Aspergillus nidulans - paving the way for efficient gene targeting. Technical University of Denmark.

[12] Galagan, J.E. et al., 2005. Sequencing of Aspergillus nidulans and comparative analysis with A. fumigatus and A. oryzae. Nature, vol. 438, no. 7971, pp.1105-15.

[13] Doonan, J.H., 1992. Cell division in Aspergillus. Journal of Cell Science, vol. 103, no. 3, pp.599-611.

[14] Nielsen, M.L. et al., 2006. Efficient PCR-based gene targeting with a recyclable marker for Aspergillus nidulans. Fungal Genetics and Biology, vol. 43, no. 1, pp.54-64.

[15] Krappmann, S., 2007. Gene targeting in filamentous fungi: the benefits of impaired repair. Fungal Biology Reviews, vol. 21, no. 1, pp.25-29.

[16] Ninomiya, Y., Suzuki, K., Ishii, C. & Inoue, H., 2004. Highly efficient gene replacements in Neurospora strains deficient for nonhomologous end-joining. PNAS, vol. 101, no. 33, pp.12248-53.

[17] Pautke, C.; Schieker, M.; TISCHER, T.; KOLK, A.; NETH, P.; Mutschler, W.; Milz, S. 2004. Characterization of Osteosarcoma Cell Lines MG-63,Saos-2 and U-2 OS in Comparison to Human Osteoblasts. Anticancer Research, vol. 24, pp. 3743-3748.