Immune System: In Vitro Study

Abstract

The immune system involves a network of specialized organs, cells and molecules that collaborate in the protection of an organism against disease. To gain more insight into the complex interactions between cells, in vitro methods and techniques have been developed to study the origin, structure and function of these organs, cells and molecules.

Keywords: lymphocytes; thymus; antibodies; DNA; cytotoxicity

Figure 1.

Schematic representation of the fetal thymic organ culture technique. The thymic lobes of a mouse embryo at day 13–14 of gestational age are explanted and treated with deoxyguanosine to remove the endogenous lymphocytes. The ‘empty’ lobes are co‐cultured with progenitor cells of mouse or human origin in a hanging drop in a Terasaki plate. The recolonized thymic lobes are subsequently cultured on top of a sponge, which allows for both liquid (medium) and gas interaction.

Figure 2.

The cloning of a deoxyribonucleic acid (DNA) fragment into a plasmid and bacterial amplification. The circular DNA plasmid contains a multiple cloning site (MCS), which is a stretch of DNA sequence specifically recognized by particular restriction enzymes (such as EcoRI and BamHI). These enzymes cut (digest) the plasmid DNA such that only DNA cut with the same restriction enzyme is able to join (ligate) together. Here the plasmid DNA and the DNA of interest to be cloned into the plasmid are cut with both the restriction enzymes BamHI and EcoRI. Subsequently the DNA fragment is inserted into the plasmid DNA and joined (ligated) by the enzyme ligase. The repaired plasmid is then introduced into the bacterium Escherichia coli (transformation) in order to amplify the plasmid with the insert DNA. The transformed bacteria are plated and grown on to an agarose solid medium plate in the presence of the antibiotic ampicillin. The ampicillin resistance gene (Amp‐r) located on the plasmid will support growth of transformed bacteria on the ampicillin‐containing medium, while outgrowth of bacteria that have not taken up plasmid DNA, and are thus not ampicillin resistant, will be prevented. Each bacterium will grow into a single colony and, as the plasmid contains an origin of replication, it will be amplified just as the bacterial chromosomal DNA with each cell division. After overnight growth, single bacterial colonies are picked and the plasmid DNA can be isolated. Digestion of the plasmid DNA with the restriction enzymes EcoRI and BamHI, and gel electrophoresis of the DNA, will confirm the presence and size of the insert DNA.

Figure 3.

The polymerase chain reaction (PCR) and reverse transcriptase‐polymerase chain reaction (RT‐PCR). The PCR reaction is performed on deoxyribonucleic acid (DNA). In the RT‐PCR reaction the starting material is ribonucleic acid (RNA), which needs to be converted to DNA before it can be amplified. The conversion of RNA to DNA is performed with the use of the enzyme reverse transcriptase which makes a complementary DNA (cDNA) strand to the messenger RNA (mRNA) strand. Oligo 2′‐deoxyribothymidine, which anneals to the poly(A) tails of mRNA, serves as a primer in this reaction. In the first cycle of the RT‐PCR, DNA is melted into single strands by increasing the temperature to 94°C. Then a DNA sequence‐specific forward primer is annealed to the template a, and the primer is elongated at 72°C by the heat‐stable DNA polymerase derived from the bacterium Thermus aquaticus. This results in template b, which consists of the complementary sequence of template a. The second cycle of the RT‐PCR, or the first cycle of a PCR reaction with DNA as starting material, starts by melting the double strands into single strands, annealing of the forward primer to template a and now also of the reverse primer to template b, and elongation of the DNA of the primers. This cycle results in templates a′ and b′. Repetition of these cycles consequently gives rise to a large amount of DNA.

Figure 4.

Generation of recombinant retrovirus. A retroviral vector is constructed that contains the complementary deoxyribonucleic acid (cDNA) encoding a gene of interest between the long terminal repeats (LTRs). These LTRs are cis‐acting elements, which mediate transcription initiation and termination. Therefore, the genetic information that is located between these LTRs is transcribed into messenger ribonucleic acid (mRNA). This vector also contains the packaging signal (ψ) required to encapsulate the viral RNA into the viral envelope (Env) proteins. The retroviral vector is transfected into a packaging signal‐deficient (ψ) host cell line. This cell line is able to transcribe and translate the retroviral gene products, Gag (translated and processed to yield virion core proteins), Pol (translated and processed to yield reverse transcriptase, integrase and a viral protease) and Env (translated and processed to yield the viral envelope glycoproteins). However, this cell line is not able to encapsulate its own viral RNA into the Env proteins because it lacks the packaging signal ψ. Only upon transfection of the ψ+ vector in the host cell line can the viral RNA be packaged; the newly formed virus particle will bud off the host cell membrane and after maturation is able to infect a target cell.

Figure 5.

Identification of the activity of a regulatory sequence by a reporter gene assay. A hypothetical regulatory sequence (promoter or enhancer sequence) is cloned into a plasmid which contains a reporter gene (e.g. the gene encoding the enzyme luciferase, or β‐galactosidase or chloramphenicol acetyltransferase). This reporter gene plasmid is transfected into a host cell where the expression of the reporter gene product is proportional to the activity of the regulatory sequence on transcription. The amount of reporter gene product can be measured by assaying its enzymatic activity in the lysates of the transfected cells. This enzymatic activity is measured in standard fluorogenic, colorimetric or chromatographic assays.

Figure 6.

Enzyme‐linked immunosorbent assay (ELISA). Antigens A and B are coated on to the surface of plastic wells to which they bind nonspecifically. An antigen A‐specific antibody, which is chemically linked to an enzyme, is added in various concentrations to the wells. Only antigen A will be specifically bound by the antibody, which causes the labelled antibody to be retained on the surface. The antigen A‐specific antibody does not bind to the unrelated antigen B. Unbound antibody is removed from the wells by washing several times. Subsequently the bound antibody is detected by an enzyme‐dependent colour change reaction and the conversion of substrate is quantified by changes in absorption, measured on a spectrometer. There are several variations on this technique which include the use of two antibodies against different epitopes of the same antigen to capture the antigen in a ‘sandwich’.

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Further Reading

Anderson G and Jenkinson EJ (1995) The role of the thymus during T‐lymphocyte development in vitro. Seminars in Immunology 7(3): 177–183.

Ausubel FM, Brent R, Kingston RE et al. (eds) (1994) Current Protocols in Molecular Biology. New York: Greene Publishing Associates.

Coligan JE, Kruisbeek AM, Margulies DH, Shevach EM and Strober W (eds) (1994) Current Protocols in Immunology. New York: Greene Publishing Associates.

Fisher AG, Larsson L, Goff LK et al. (1990) Human thymocyte development in mouse organ cultures. International Immunology 2(6): 571–578.

Jenkinson EJ and Anderson G (1994) Fetal thymic organ cultures. Current Opinion in Immunology 6: 293–297.

Jenkinson EJ, Franchi LL, Kingston R and Owen JJT (1982) Effect of deoxyguanosine on lymphopoiesis in the developing thymus rudiment in vitro: application in the production of chimeric thymus rudiments. European Journal of Immunology 12: 583–587.

Oosterwegel MA, Haks MC, Jeffry U, Murray R and Kruisbeek AM (1997) Induction of TCR gene rearrangements in uncommitted stem cells by a subset of IL‐7 producing, MHC class‐II‐expressing thymic stromal cells. Immunity 6(3): 351–360.

Roncarolo MG, Carballido JM, Rouleau M, Namikawa R and de Vries JE (1996) Human T‐ and B‐cell functions in SCID‐hu mice. Seminars in Immunology 8(4): 207–213.

Sambrook J, Fritsch EF and Maniatis T (eds) (1989) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press.

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How to Cite close
Blom, Bianca, and de Waal Malefyt, René(Apr 2001) Immune System: In Vitro Study. In: eLS. John Wiley & Sons Ltd, Chichester. http://www.els.net [doi: 10.1038/npg.els.0001130]