Mammalian cells

Mammalian cells are higher eukaryotic cells derived from multicellular organisms. Eukaryotic cells have a unique ability to process proteins post-translationally, and they contain a large number of membrane bound compartments such as mitochondria, endoplasmatic reticulum, the Golgi apparatus. Compared to microbes, mammalian cells are fragile, have a slow doubling time (app. 24h), and need complex growth media. The cells are also easily contaminated with mycoplasma, so it is necessary to work completely sterile, when handling mammalian cells(1). So what's the deal with these high maintenance cells? Microbial cells are excellent for production of different compounds like peptides and simple proteins such as insulin and growth hormones. Why working with mammalian cells at all?

Mammalian cell factories

Mammalian cell cultures represent a suitable and stabile gene expression system and are often used as cell factories for production of biopharmaceuticals. Heterologous protein expression in a suitable host is central in production of biopharmaceuticals, therefore mammalian cell cultures are widely used for production of therapeutic proteins such as monoclonal antibodies, growth hormones, and cytokines used for a wide array of diseases(4). 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). The effect of post-translationally modifications are protein stability, proper ligand binding, and reduced risk of immunogenicity (2). Most of the therapeutic proteins approved and currently in development are post-translationally 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. The original cells were taken from bone tissue of the tibia of a 15 year old girl suffering from osteosarcoma. An immortalized cell line has acquired 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. In comparison, the HeLa cell line contain the well known HPV virus. They are also very good-looking in a confocal microscope, and therefore U-2 OS was chosen for proof of concept of Plug 'n’ Play in mammalian cells.

Transient transfection

Transient expression is the ability to express a 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 protein characterization or to verify the integrity, functionality, and the efficiency of different recombinant vectors. Production of large amount of recombinant protein has been reported for transient expression system on 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).

The mammalian expression vectors have a multiple cloning site (MCS). The gene of interest is therefore required to hold restriction sites compatible with the expression vector and the insertion of the gene is achieved by ligase. The method is often cumbersome in construction of the expression vector. Furthermore, the integration of the gene of interest in the expression vector by restriction enzymes and ligases has low efficiency as well as provide high number of false-positive results(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. Some of the filamentous fungal species are pathogenic to humans, whereas others have great value in the production of antibiotics such as penicillin. The fungi are therefore importance for industrial as well as medical production.
Filamentous fungi produce a diverse array of secondary metabolites, which serve the pharmaceutical sciences as prolific source of chemical compounds for the development of new drugs.

Growth of filamentous fungi

The vegetative growth of filamentous fungi start with the germination of a spore when the conditions are right . The spore germination leads to formation of hyphae (7). A fungal hypha 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 (9).

Filamentous fungi grow by the polar extension of hyphae and multiply by branching (10). The branched hyphae forms a network of interconnected cells called a mycelium (7). The mycelium forms a radially symmetric colony that expands over 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 and development (7).

Aspergillus nidulans

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

The genetic analysis has produced a deep understanding of both the physiology of Aspergillus and the organisation of the genome (13). This research has advanced the study of eukaryotic cellular physiology and contributed to our understanding of metabolic regulation, development, DNA repair, morphogenesis, and human genetics 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

Accurate manipulation of genes is a key approach in fungal molecular biology (15). The method of gene targeting facilitates precise genome manipulations which are called site directed alterations. They are performed by use of deletions, replacements and insertion in the 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. Different DNA fragments are constructed depending on deletion, replacement or insertion. The fungi can integrates linear DNA fragment in its genome by using its repair system of DNA double stranded breaks.

Two mechanisms of DNA double strand break repair ensure that the introduced piece of DNA is pasted into the fungal genome, to be replicated stably; Homologous Recombination and Non Homologous End Joining, also called illegitimate recombination (15). Homologous recombination involves interaction between homologous sequences, whereas Non Homologous End Joining involves ligation of the strand ends independently of DNA homology (16). Precise genome manipulation can often be tedious and time-consuming, because fungi appear to favour Non Homologous End Joining over Homologous recombination 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.