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Proof of concept in mammalian cells

Mammalian cells

Mammalian cells are higher eukaryotic cells derived from multicellular organisms. These cells contain a large number of membrane bound compartments like mitochondria, endoplasmatic reticulum, the Golgi apparatus etc. Eukaryotic cells also have a unique ability to process proteins post-translationally. Compared to microbes, mammalian cells are fragile, have a slow doubling time (app. 24h),require complex media and sophisticated fermentation setups for production processes (Mueller et al., 2003). Besides, microbial cells are excellent for production of different compounds like peptides and simple proteins such as insulin and growth hormones. So why use mammalian cells for industrial production at all?

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.(Xie, Zhou, & Robinson, 2003) 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 (Mueller et al., 2003). The effect of post-translationally modifications are protein stability, proper ligand binding, and the risk of immunogenicity is also reduced (4). Most of the therapeutic proteins approved and currently in development are post-translationally modified (5,5). 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 (Wurm, 2004).

Growth

Lots of different mammalian cell lines have been derived.Mammalian cells can grow in suspension or as adherent cells They require complex media

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 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 (Bollati-Fogolín & Comini, 2008).
The success of transfections depends on several factors that must be taken intoaccount:

1. the transfectability and physiology of the cell line

2. the type of expression desired

3. the genetic marker in the expression vector

4. the size of the expression cassette and the quality of the DNA introduced

5. the compatibility of transfection method and the cell line

6. the use of assay for detection of recombinant protein

7. the presence of serum and/or antibiotics in the culture medium

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 achieve 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 have low efficiency as well as provide high number of false-positive (Bollati-Fogolín & Comini, 2008).

Proof of concept

To demonstrate how fast any vector of choice for the expression in mammalian cells can be assembled we chose to design a reporter system as proof of concept. This system comprises a backbone plasmid that allows for the amplification in and selection of E. coli, the strong constitutive cytomegalovirus (CMV) promoter, a gene module consisting of a gene encoding a fluorescence protein or a gene encoding a fluorescence protein and a localization signal, the BGH terminator and the hygromycin marker cassette. This reporter system can easily be modified and used for gene expression studies. Furthermore we designed a vector for localization of GFP to the peroxisomes, which for instance could be used to monitor movement and metabolism. The idea is to develop this reporter system so that proteins can be localized to organelles and other subcellular compartments allowing for the study of organelles in real time. We also showed that cells can be transfected with different fluorescence proteins at the same time. Further development of this system would allow targeting of different compartments with different fluorescence proteins at the same time. The U-2 OS cells were transiently transfected with the different plasmids constructed. The cells were fixed using 4% formaldehyde, and VECTASHIELD was added to prevent rapid loss of fluorescence while examining the cells microscopically. Visualization was performed with a confocal microscope.

pJEJAM1 BBa_K678049

BBa_K678049 is a plasmid intended for transient transfection of mammalian cells. The expression of the green fluorescence protein is under the control of the strong constitutive CMV promoter, which can be seen in the figure below.


U-2 OS cells transiently transfected with plasmid BBa_K678049 expressing GFP. This vector provides a good expression and homogenous distribution of GFP. The white bar has a length of 20μm.	U-2 OS cells transiently transfected with pJEJAM1 expressing GFP. This vector provides a good expression and homogenous distribution of GFP. The white bar has a length of 20μm.

pJEJAM2 BBa_K678050

BBa_K678050 is a plasmid intended for transient transfection of mammalian cells. The expression of the green fluorescence protein (GFP) is under the control of the strong constitutive CMV promoter (see the figure below. The peroxisomal targeting signal PTS1 is directly fused to the C-terminal of GFP, this sequence ensures the localization of GFP to the peroxisomes of the cell.

U-2 OS cells transiently transfected with plasmid BBa_K678050 expressing GFP localized to the peroxisomes. As can be seen on the picture this vector as intended localizes GFP to the peroxisomes of the cells. The white bar has a length of 30μm.

pJEJAM3 BBa_K678051

BBa_K678051 is a plasmid intended for transient transfection of mammalian cells. The expression of the yellow fluorescence protein (YFP), is under the control of the strong constitutive CMV promoter (see the figure below).


U-2 OS cells transiently transfected with plasmid BBa_K678051 expressing YFP. This vector provides a good expression and homogenous distribution of YFP. The white bar has a length of 40μm.	U-2 OS cells transiently transfected with plasmid BBa_K678051 expressing YFP. This vector provides a good expression and homogenous distribution of YFP. The white bar has a length of 30μm.

pJEJAM4 BBa_K678052

BBa_K678052 is a plasmid intended for transient transfection of mammalian cells. The expression of mCherry, a red fluorescence protein, is under the control of the strong constitutive CMV promoter (see the figure below).


U-2 OS cells transiently transfected with plasmid BBa_K678052 expressing mCherry. This vector provides a good expression and homogenous distribution of mCherry. The white bar has a length of 70μm.	U-2 OS cells transiently transfected with plasmid BBa_K678052 expressing mCherry. This vector provides a good expression and homogenous distribution of mCherry. The white bar has a length of 50μm.

pJEJAM5 BBa_K678053

BBa_K678053 is a plasmid intended for transient transfection of mammalian cells. The expression of cyan fluorescence protein (CFP), is under the control of the strong constitutive CMV promoter (see the figure below).


U-2 OS cells transiently transfected with plasmid BBa_K678053 expressing CFP. This vector provides a good expression and homogenous distribution of CFP. The white bar has a length of 70μm.	U-2 OS cells transiently transfected with plasmid pJEJAM5 expressing CFP. This vector provides a good expression and homogenous distribution of CFP. The white bar has a length of 50μm.

Play'n'Mix

Mammalian cells can be transiently transfected with several plasmids at once, allowing the simultaneous expression of different fluorescence proteins. The pictures below demonstrate different combinations of fluorescent proteins.


U-2 OS cells transiently transfected with plasmids BBa_K678050 and BBa_K678053 expressing GFP localized to the peroxisomes and CFP. The white bar has a length of 30μm.	U-2 OS cells transiently transfected with plasmids BBa_K678049 and BBa_K678053 expressing GFP and CFP. The white bar has a length of 30μm.

U-2 OS cells transiently transfected with plasmids BBa_K678049 and BBa_K678052 expressing GFP and mCherry. The white bar has a length of 70μm.	U-2 OS cells transiently transfected with plasmids BBa_K678052 and BBa_K678053 expressing YFP and CFP. The white bar has a length of 40μm.

References

(1) Hesse, F., Wagner, R. (2000). Developments and improvements in the manufacturing of human therapeutics with mammalian cell cultures. TIBTECH, 18, 173-180.

(2) Global Biopharmaceutical Market Report (2010-2015). (2010). The International Market Analysis Research and Consulting Group.

(3) 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.

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

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

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