Gene Expression in Mammalian Cells and its Applications

Mammalian protein expression system has the function of protein folding and post-translational modification which let the protein closer to the natural protein, so that to obtain the same biological activity with natural protein. Therefore, the Mammalian Cell Expression System is the most widely used in the development and production of recombinant protein drugs, particularly in the therapeutic monoclonal antibodies.

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The production of proteins in appropriate quantity and quality is an essential requirement of the present time. There appears to be a progressive increase in the application of mammalian cells for proteins production. Expression systems utilizing mammalian cells for recombinant proteins are able to introduce proper protein folding, post-translational modifications, and product assembly, which are important for complete biological activity. This review article is totally based on literature survey. In this article much emphasis has been done on the mammalian expression system. The author focused on different mammalian cell lines that express the gene. The different vector systems that transfer the gene into mammalian cells like plasmid based expression vectors, adenovirus vectors, vaccinia vectors, retroviral vector and baculovirus as vectors were explored. The processes for the transfer of gene into mammalian cells were also reviewed. Application and limitations of mammalian expression system were also focused. The purpose of research in writing this article is to create awareness in researchers, starting their career in gene expression related to mammalian cells. The principal result and major conclusion of this article is to make available the molecular technologies, expression system and applications of gene expression in mammalian cell lines. Introduction Gene is defined as a discrete unit of genetic information which is required for the production of a polypeptide. It includes the coding sequence, the promoter and terminator, and introns. Expression can be described as a transcription and translation of a gene. A particular host is required to express a particular gene. Today, there are wide selections of expression systems available for large-scale recombinant protein production. These expression systems include E. coli, baculovirus-mediated insect cell expression, yeast, and several mammalian based systems. Each has its own respective advantages in relation to cost, ease of use, and their post-translational modification profiles. Factors to be considered while considering an expression system for gene expression are listed in Table 1. This author reported earlier in his articles about gene transfer technology.1-3 In this article the author reviewed the mammalian expression system. The introduction of large-scale transient transfection has enabled the use of mammalian cells more attractive in terms of speed and ease of use, particularly for cell surface and secreted glycoproteins. The features of the transient expression systems have been listed in the Table 2. As compared to insect cells, glycosylation in mammalian cells results in attachment of large and complex glycans to the ex-pressed proteins which generally interfere with crystallization. However, strategies are available that make the glycosylation to be modified. This has led to the successful crystallization of glycoproteins produced in mammalian cells.4 Reports were also made regarding the protein expression in mammalian cells.5 This article is totally based on literature survey. The author considered the modern as well as traditional method of literature survey. The search engine and data bases utilized were Science direct, PubMed and Google. The literatures cited in this article are not limited to a particular region but is considered more or less from the entire globe. The author tried to more on the recent research related to the topic. Literatures for the past five years including the year 2012 and valuable literatures of the past were also considered in making this manuscript. The main objective of this work is to review the different mammalian cell lines that are used for transfection that finally leads to gene expression. The author reviewed the vectors used in transfection in mammalian cell lines. The author also described the various method of gene transfer in mammalian cells. Much stress has been given on the application of gene expression in mammalian cell line. The aim of this article is to create awareness in young researchers whose aim is to express genes in mammalian cell lines to get the desired protein. This article will be beneficial for readers who are initially starting their carrier in biotechnology through animal tissue culture and gene expression. Requirements for mammalian expression system The expression level of a gene largely depends upon how efficiently it is transcribed. Transcription of any gene takes place when the RNA polymerase complex interacts with the promoter sequences moves along the gene from a 5ı to 3ı direction. This produces an RNA transcript and finally dissociating from the gene at the transcription signal, freezing the transcript for eventual translation. The gene expression in mammalian cells needs a suitable cell line and the appropriate vectors that should act as a vehicle to transport the gene of required interest into the required cell lines. Cell lines In the previous decade, protein therapeutics produced from mammalian cells have changed the landscape of human healthcare. The value of protein therapeutics has made the search in the forward direction for more costeffective and efficient cell lines that are capable of producing high quality protein products. Bioprocesses based on mammalian cell have been applied in the manufacture of viral vaccines, diagnostic and therapeutic proteins in the past. In the production of protein therapeutics, cells are the host for producing proteins. The most widely used host mammalian cells are chinese hamster ovary (CHO) cells and mouse myeloma cells, including NS0 and Sp2/0 cells.6 Two derivatives of the CHO cell line, CHO-K1 and CHO pro-3, gave rise to the two most commonly used cell lines in bioprocessing today, DUKX-X11 and DG44. These two cell lines were engineered to be deficient in dihydrofolate reductase (DHFR) activity.7-8 A number of mammalian cell lines have been utilized for protein expression with the most common being HEK 293 (Human embryonic kidney) and CHO (Chinese hamster ovary). These cell lines can be transfected using polyethyleneimine (PEI) or calcium phosphate. HEK 293 cells exhibit the highest level of PEI-mediated transfection with 50–80% of cells showing green fluorescent protein (GFP) expression,9 and are now widely used for production of recombinant proteins both by transient transfection as well as by the formation of stable cell lines. Protein expression in mammalian cells can also be achieved using viral-mediated transduction by such techniques as the BacMam system.10 This technology utilizes recombinant baculoviruses for simple transduction of mammalian cells, allowing for production of milligram quantities of protein for structural studies.11 Other cell lines such as COS and Vero (both green African monkey kidney), HeLa (Human cervical cancer), and NS0 (Mouse myeloma) have also been used for structural studies. Some of these cell lines such as NS0 are more difficult to transfect. Transfection can be usually achieved using electroporation, and are only used in stable cell line production. A number of mammalian cell lines have been summarized in Table 3.