Team:DTU-Denmark-2/results/Proofofconcept/mammalian

Why mammalian cells?

The potential in biopharmaceuticals produced by mammalian cells is huge (1). Last year the global biopharmaceutical market in 2015 was expected to increase to US$ 167 billion (2). Biopharmaceuticals target diseases such as psoriasis and cancer and are more convenient, safe and effective than the other treatment solutions on the market (3). Compared to microbes mammalian cells have lower yields, are more difficult to handle in the laboratory, are costly and have a slow doubling time. So why use mammalian cells when microbes such as yeast and E. coli are already used as fast, efficient, and cheap cell factories?
The main and most important answer to this is post-translational modifications (PTMs) (4). The reasons for the importance of the PTMs are the protein stability, ligand binding, and potential result in increased immunogenicity when used for humans (4). Most of the therapeutic proteins approved and currently in development are post-translationally modified, which is not surprising since approximately 50% of human proteins are glycosylated (5,5).

With this kept in mind there are some key issues in manufacturing where speed in development plays a prominent role; cell line creation has to be completed as rapidly as possible. To reduce the timelines for cell line creation the Plug’n’Play with DNA assembly standard could be a valuable tool.

It is not only in the industry a more efficient high throughput cloning system could be valuable, when it comes to constructing DNA libraries from extremely complex DNA populations such as total human genomic DNA the application of the Plug'n'Play standard could reduce the construction time with several weeks.

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 fluorescent protein or a gene encoding a fluorescent 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 lastly showed that cells can be transfected with different fluorescent proteins which with a further development of the system would allow labeling with different fluorescent proteins.

pJEJAM1

pJEJAM1 is a plasmid intended for transient transfection of mammalian cells. The expression of the green fluorescent 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 pJEJAM1 expressing GFP. This vector provides a good expression and homogenous distribution of GFP. Cells were fixed using 4% formaldehyde and addition of VECTASHIELD. Imaging was performed with a confocal microscope, 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. Cells were fixed using 4% formaldehyde and addition of VECTASHIELD. Imaging was performed with a confocal microscope, the white bar has a length of 20μm.

pJEJAM2

pJEJAM2 is a plasmid intended for transient transfection of mammalian cells. The expression of the green fluorescent 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 pJEJAM2 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. Cells were fixed using 4% formaldehyde and addition of VECTASHIELD. Imaging was performed with a confocal microscope, the white bar has a length of 30μm.

pJEJAM3

pJEJAM3 is a plasmid intended for transient transfection of mammalian cells. The expression of the yellow fluorescent protein (YFP), is under the control of the strong constitutive CMV promoter (see the figure below).

U-2 OS cells transiently transfected with plasmid pJEJAM3 expressing YFP. This vector provides a good expression and homogenous distribution of YFP. Cells were fixed using 4% formaldehyde and addition of VECTASHIELD. Imaging was performed with a confocal microscope, the white bar has a length of 40μm.	U-2 OS cells transiently transfected with plasmid pJEJAM3 expressing YFP. This vector provides a good expression and homogenous distribution of YFP. Cells were fixed using 4% formaldehyde and addition of VECTASHIELD. Imaging was performed with a confocal microscope, the white bar has a length of 30μm.

pJEJAM4

pJEJAM4 is a plasmid intended for transient transfection of mammalian cells. The expression of mCherry, a red fluorescent protein, is under the control of the strong constitutive CMV promoter (see the figure below).

U-2 OS cells transiently transfected with plasmid pJEJAM4 expressing mCherry. This vector provides a good expression and homogenous distribution of mCherry. Cells were fixed using 4% formaldehyde and addition of VECTASHIELD. Imaging was performed with a confocal microscope, the white bar has a length of 70μm.	U-2 OS cells transiently transfected with plasmid pJEJAM4 expressing mCherry. This vector provides a good expression and homogenous distribution of mCherry. Cells were fixed using 4% formaldehyde and addition of VECTASHIELD. Imaging was performed with a confocal microscope, the white bar has a length of 50μm.

pJEJAM5

pJEJAM5 is a plasmid intended for transient transfection of mammalian cells. The expression of cyan fluorescent protein (CFP), is under the control of the strong constitutive CMV promoter (see the figure below).

U-2 OS cells transiently transfected with plasmid pJEJAM5 expressing CFP. This vector provides a good expression and homogenous distribution of CFP. Cells were fixed using 4% formaldehyde and addition of VECTASHIELD. Imaging was performed with a confocal microscope, 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. Cells were fixed using 4% formaldehyde and addition of VECTASHIELD. Imaging was performed with a confocal microscope, 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 fluorescent proteins. The pictures below demonstrate different combinations of fluorescent proteins.

U-2 OS cells transiently transfected with plasmids pJEJAM2 and pJEJAM5 expressing GFP localized to the peroxisomes and CFP, respectively. Cells were fixed using 4% formaldehyde and addition of VECTASHIELD. Imaging was performed with a confocal microscope, the white bar has a length of 30μm.	U-2 OS cells transiently transfected with plasmids pJEJAM1 and pJEJAM5 expressing GFP and CFP, respectively. Cells were fixed using 4% formaldehyde and addition of VECTASHIELD. Imaging was performed with a confocal microscope, the white bar has a length of 30μm.
U-2 OS cells transiently transfected with plasmids pJEJAM1 and pJEJAM4 expressing GFP and mCherry, respectively. Cells were fixed using 4% formaldehyde and addition of VECTASHIELD. Imaging was performed with a confocal microscope, the white bar has a length of 70μm.	U-2 OS cells transiently transfected with plasmids pJEJAM4 and pJEJAM5 expressing YFP and CFP, respectively. Cells were fixed using 4% formaldehyde and addition of VECTASHIELD. Imaging was performed with a confocal microscope, the white bar has a length of 40μm.

(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.