Team:DTU-Denmark-2/results/background
From 2011.igem.org
(Difference between revisions)
(One intermediate revision not shown) | |||
Line 87: | Line 87: | ||
<p align="justify"> | <p align="justify"> | ||
- | Mammalian cells are | + | 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? </p> |
<br> | <br> | ||
Line 93: | Line 93: | ||
<p align="justify"> | <p align="justify"> | ||
- | 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 | + | 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). </p> | 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). </p> | ||
<br> | <br> | ||
Line 100: | Line 100: | ||
<p align="justify"> | <p align="justify"> | ||
- | 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 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. </p> |
<br> | <br> | ||
Line 116: | Line 116: | ||
<dd>7. the presence of serum and/or antibiotics in the culture medium</dd> | <dd>7. the presence of serum and/or antibiotics in the culture medium</dd> | ||
<p align="justify"> | <p align="justify"> | ||
- | 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 | + | 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). </p> |
<br><br> | <br><br> | ||
Line 122: | Line 122: | ||
<a name="Filamentous Fungi"></a><h1><b>Filamentous Fungi</b></h1> | <a name="Filamentous Fungi"></a><h1><b>Filamentous Fungi</b></h1> | ||
<p align="justify"> | <p align="justify"> | ||
- | 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 | + | 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 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 great value in the production of antibiotics such as penicillin. Fungi are therefore of great importance for the industry as well as for our daily life. <br> |
</p><br> | </p><br> | ||
<a name="Growth of filamentous fungi"></a><h3><b>Growth of filamentous fungi</b></h3> | <a name="Growth of filamentous fungi"></a><h3><b>Growth of filamentous fungi</b></h3> | ||
<p align="justify"> | <p align="justify"> | ||
- | + | Under the right conditions, the vegetative growth of filamentous fungi starts 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). </p> | |
<p align="center"> | <p align="center"> | ||
Line 139: | Line 139: | ||
<a name="Aspergillus nidulans"></a><h3><b><i>Aspergillus nidulans</i></b></h3> | <a name="Aspergillus nidulans"></a><h3><b><i>Aspergillus nidulans</i></b></h3> | ||
<p align="justify"> | <p align="justify"> | ||
- | The filamentous fungus <i>Aspergillus nidulans</i> is a model organism and | + | The filamentous fungus <i>Aspergillus nidulans</i> is a model organism, and in contrast to most other aspergilla it has a well characterized sexual cycle and a well-developed genetic tools for manipulation (9,12). Furthermore, in <i>A. nidulans</i> the parasexual cycle has been extensively utilized. Parasexual genetics involves examination of recombination in the absence of sexual reproduction.<br> |
<br> | <br> | ||
The genetic analysis has produced a deep understanding of both the physiology of <i>Aspergilli</i> 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 <i>A. nidulans </i>has created a tremendous potential to obtain insight into important aspects of fungal biology such as transcriptional regulation, secondary metabolite production and pathogenicity (14). </p> | The genetic analysis has produced a deep understanding of both the physiology of <i>Aspergilli</i> 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 <i>A. nidulans </i>has created a tremendous potential to obtain insight into important aspects of fungal biology such as transcriptional regulation, secondary metabolite production and pathogenicity (14). </p> | ||
Line 214: | Line 214: | ||
[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. | [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. | ||
<br><br> | <br><br> | ||
+ | |||
+ | [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. | ||
Latest revision as of 14:44, 21 September 2011
Background