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Immunization (Generation of antibodies)

An antigen can be almost any molecular substructure (i.e. part of a molecule). Antibodies are proteins, which specifically bind to an antigen and are produced by specialized white blood cells (B-cells) of the immune system upon contact with foreign (i.e. not self) antigens.

This defense mechanism against pathogens can be exploited to produce antibodies against any molecule of interest. Repetitive immunization with an antigen will generate an immune response and lead to the production of many B-cells that produce vast amount of antibodies directed against the antigen. These antibodies can be used in the laboratory to detect specific antigens (e.g. pregnancy test).

The serum (i.e. antiserum) of the blood of the immunized animal contains the antibodies, which can be used for many different techniques. Antisera are often called polyclonal antibodies.

It is also possible to isolate the antibody-producing B-cells from the animals to fuse them with a cancer cell line. The cell hybrids are called are called hybridoma. The technique also allows selection of successfully fused B-cells, which produce the antibody of interest, and indefinite expansion in cell culture. As the hybridoma secretes its antibody into the extracellular environment, the cell culture supernatant contains the antibody of interest. Because a hybridoma secrets one type of antibody originating from one B-cell, these antibody preparations are called monoclonal antibodies.

Immunofluorescence – IF

Immunofluorescence is a method to visualize antigens of cells by using antibodies.

Antibodies can be used to probe cells to test whether the antigen is present. The cells can be freshly isolated primary cells or cells in cell culture. Specific antibodies that detect the antigen of interest can be generated by immunization. To visualize the antibodies bound to the antigen, the antibody can be labeled in various ways. Most common are fluorophores or enzymes as labels. Usually a so-called secondary antibody is labeled, which detects the primary antibodies bound to the antigen of interest. This results in a sandwich composed of the antigen that is bound by the primary antibody, which is in turn bound by the secondary antibody that carries the label for detection. Fluorescent labels can be detected using fluorescence microscopes and enzymes as label can be detected by their enzymatic activity, which is a color reaction.

Next Generation Sequencing – NGS

NGS is a method that allows the sequencing of millions of DNA templates in parallel in a cost-efficient and fast manner.

Because many different NGS protocols from different companies are around and due to the complexity of the sample preparation and sequencing process, only one widely used NGS system (Illumina®) is explained here.

The DNA of interest is first chopped in to smaller pieces of a relatively uniformed size and are ligated with adaptors to which the primers will bind. These fragments (the library) are attached to a solid carrier for example a glass slide. The billons of different DNA fragments are then clonally amplified on the carrier. Sequencing is performed in real-time by adding a DNA polymerase and reversibly fluorescently labeled nucleotides. For each base of the millions of different templates that is to be sequenced, the polymerase will add the respective nucleotide. Then the whole glass slide is excited by laser light, which will lead to a fluorescent signal for each newly added base of each fragment (as for Sanger sequencing, the four bases again have four different colors) and a picture is taken. Then the label is removed and the process is repeated many times. The resulting reads are between 50 and 250 base pairs. The total amount of sequence is around some billions of base pairs, which are highly redundant. The reads are assembled usually using a reference sequence to which they are mapped.

RNA interference (RNAi)

RNAi is a method to specifically inhibit the expression of genes at the mRNA level by short doublestranded RNA molecules complementary to the gene of interest, which govern the degradation of the respective mRNA.

RNA interference was discovered by Andrew Fire and Craig Mellow in 1998, which received the Nobel Prize Medicine for this groundbreaking work. They discovered that introduction of doublestranded RNA (dsRNA) led to specific degradation of mRNA that match the sequence of the dsRNA in the roundworm C. elegans. This pathway is conserved in many eukaryotes and represent an additional level of gene expression regulation.

The cell itself expresses so called micro-RNAs (miRNAs) that target and silence the expression of groups of genes. Exogenous dsRNAs are usually introduce experimentally and are designed to knock down specifically the gene of interest. Note that miRNA transcripts do not encode for proteins.

Both types of RNAs are processed by an enzyme called Dicer, a ribonuclease, which cuts the RNAs into short fragments of 20 to 25 nucleotides in length, which are called short interfering RNAs (siRNAs). The strands of the siRNAs are separated and are integrated in an enzymatic complex named RNA-induced-silencing-complex (RISC). The RISC complex scans mRNAs for complementarity to the siRNA strand and if complementarity is detected, endonucleases of the Argonaut protein family degrade the mRNA preventing thereby expression of the protein.

qPCR – quantitative reverse-transcription polymerase-chain-reaction

The method is based on three more simple techniques, which are polymerase chain reaction (see PCR), reverse transcription and detection of DNA by fluorescent signals.

Reverse transcription is the process that converts RNA into single stranded DNA, a so called copy DNA (cDNA). A reverse transcriptase is an enzyme with a RNA-dependent DNA polymerase activity and is widely used to convert mRNAs (messenger RNA, protein coding) into cDNA. Because mRNAs have a long stretch of adenines at their 3’-end (poly-A), they are often reverse transcribed using a primer composed of a long stretch of thymindines (poly-T). Thus a cDNA-preparation often contains the totality of all protein-coding mRNAs from the tissue or cell type from which the RNA was isolated.

There is also a large number of non-coding transcripts, which are as important for a cell as the protein-coding mRNAs. The most prominent examples are ribosomal and transfer RNAs that are crucial for protein synthesis. Another prominent member of these transcripts are micro RNAs, which are only known since 15 years, that mediate posttranslational control of gene expression.

Quantitative reverse transcription PCR is a highly sensitive, quantitative method that allows the quantification of expression levels of RNAs (i.e. how much is there in a cell). qPCR simply detects the amount of DNA generated in each PCR cycle by fluorescent signals. There are several technical variants to detect DNA. The most simple form is to add a fluorescent dye to the PCR reaction, which can only bind double stranded DNA but not to nucleotides or single stranded DNA (such as primers or cDNA). The fluorescence is directly measured in the reaction plate in the qPCR machine.

Microarray

A microarray is a technique to detect changes in gene expression of thousands of genes in parallel.

Microarrays are usually used to analyze the transcriptome of a sample. The transcriptome of an organism is the entirety of all genes expressed during its life time including embryogenesis. A microarray relies on specific probes, which are short pieces of chemically synthetized DNA complementary to the gene of interest. For each gene present on the microarray several little spots with the probe are attached to a slide. That means if the transcriptome of an organism is about 25.000 mRNA, the array would have about 100.000 spots in total, if four spot are printed per gene.

To analyze the transcriptome of a sample, the RNA needs to be isolated, reverse transcribed into cDNA (see also qPCR) and labeled fluorescently. Because the probe can bind extremely specifically to its target mRNA, the fluorescent labeled cDNA can now be hybridized to the microarray. After binding, the microarray can be washed and imaged using an automated fluorescent microscope. Because each spot contains some billions of probe molecules, the fluorescent signal intensity does not only tell whether a gene is expressed but also how much. Because each potentially expressed gene is present as probe on the microarray, the activity of virtually all genes in the sample compared to the control can be accessed at once.

The signals are then normalized to controls such as house keeping genes, which allows quantification and comparison of different samples. Microarrays have been proven to be an extremely versatility and useful tool in molecular biology.

Fluorescence in situ hybridization (FISH)

FISH is a method to detect expression of RNAs in cells of tissues with high spatial resolution.

FISH exploits the property of single-stranded RNA/DNA molecules to form double-strands with respective complementary RNA/DNA molecules. Often RNA probes are used to detect specific transcripts.

To investigate the expression of an gene of interest in a tissue of interest, the gene has to be cloned into a vector that contains a RNA polymerase promoter. Alternatively, the RNA-polymerase promoter can added to the gene directly by a PCR reaction.

The RNA-probe is generated by in vitro translation, which yields RNA molecules complementary to the RNA of interest. In addition, some of the nucleotides are labeled usually with digoxigenin. The RNA-probe can then be incubated with a section of the tissue or cells of interest and it will bind to the respective RNA. The label can then be detected using antibodies, which are in turn labeled fluorescenct dye. After washing away free-probe, the expression of the respective RNAs can be visualized by fluorescence microscopy.

Cell culture

Cell culture is a method to culture cell lines or primary cells in vitro. Cells in cell culture are accessible and easy to manipulate making experimental setups convenient.

The basic principles of cell culture were already established beginning of the 20th century, when developmental biologists started to culture organs and embryo explants in salt solutions supplemented with body fluids such as amniotic fluid, allantois extract or serum. In the 50ties of the previous century the widespread virology research spurred the development of robust cell culture protocols for mammalian cell lines, which were used to produce viral particles for research and vaccine development.

Cells freshly isolated from the body are called primary cells. The cells that can be isolated most easily from mammals are cells of the peripheral blood. The isolation of cells from tumors and subsequent long term culture or infection of primary cells with tumorigenic viruses lead to the generation of many cell lines, which are virtually immortal and can be cultured much easier than primary cells.

Establishing cell culture conditions was and is still trail and error by just testing out many different ingredients and conditions, and is usually very complex. For a long time the biological reasons for many ingredients that support the growth of a particular cell type were unclear or as for serum the active ingredients were just unknown. However, since the 1950ies we learned a lot about a cells biology and understand much better what a cell need to be happy. Yet, out of the approximate 200 different cell types of the human body only some can be cultured with ease. To reduce experimental variability, there are a lot of efforts to get rid of variable and undefined ingredients such as serum and instead to move towards chemically defined medias in which the concentration of all components, ranging from salts, organic chemicals, amino acids, peptides and proteins, are exactly known

But if you are a scientist nowadays working with mammalian cells, cell culture is in fact a routine job. Basically all media, supplements and dishes can be purchased commercially. For all cell lines and many primary cells robust standard culturing protocols are available.