CD8 is a transmembrane glycoprotein that serves as a co-receptor for the T cell receptor. Like the TCR, CD8 binds to a major histocompatibility complex molecule, but is specific for the class I MHC protein. There are two isoforms of the protein and beta, each encoded by a different gene. In humans, both genes are located on chromosome 2 in position 2p12; the CD8 co-receptor is predominantly expressed on the surface of cytotoxic T cells, but can be found on natural killer cells, cortical thymocytes, dendritic cells. The CD8 molecule is a marker for cytotoxic T cell population, it is expressed in T cell lymphoblastic lymphoma and hypo-pigmented mycosis fungoides. To function, CD8 forms a dimer; the most common form of CD8 is composed of a CD8-α and CD8-β chain, both members of the immunoglobulin superfamily with an immunoglobulin variable -like extracellular domain connected to the membrane by a thin stalk, an intracellular tail. Less-common homodimers of the CD8-α chain are expressed on some cells.
The molecular weight of each CD8 chain is about 34 kDa. The structure of the CD8 molecule was determined by Leahy, D. J. Axel, R. and Hendrickson, W. A. by X-ray Diffraction at a 2.6A resolution. The structure was determined to have an immunoglobulin-like beta-sandwich folding and 114 amino acid residues. 2% of the protein is wound into α-helices and 46% into β-sheets, with the remaining 52% of the molecules remaining in the loop portions. The extracellular IgV-like domain of CD8-α interacts with the α3 portion of the Class I MHC molecule; this affinity keeps the T cell receptor of the cytotoxic T cell and the target cell bound together during antigen-specific activation. Cytotoxic T cells with CD8 surface protein are called CD8+ T cells; the main recognition site is a flexible loop at the α3 domain of an MHC molecule. This was discovered by doing mutational analyses; the flexible α3 domain is located between residues 229 in the genome. In addition to aiding with cytotoxic T cell antigen interactions the CD8 co-receptor plays a role in T cell signaling.
The cytoplasmic tails of the CD8 co-receptor interact with Lck. Once the T cell receptor binds its specific antigen Lck phosphorylates the cytoplasmic CD3 and ζ-chains of the TCR complex which initiates a cascade of phosphorylation leading to activation of transcription factors like NFAT, NF-κB, AP-1 which affect the expression of certain genes. T-cell Group - Cardiff University Mouse CD Antigen Chart Human CD Antigen Chart CD8 alpha - Marker for cytotoxic T lymphocytes
The T-cell receptor, or TCR, is a molecule found on the surface of T cells, or T lymphocytes, responsible for recognizing fragments of antigen as peptides bound to major histocompatibility complex molecules. The binding between TCR and antigen peptides is of low affinity and is degenerate: that is, many TCRs recognize the same antigen peptide and many antigen peptides are recognized by the same TCR; the TCR is composed of two different protein chains. In humans, in 95% of T cells the TCR consists of an alpha chain and a beta chain, whereas in 5% of T cells the TCR consists of gamma and delta chains; this ratio changes in diseased states. It differs between species. Orthologues of the 4 loci have been mapped in various species; each locus can produce a variety of polypeptides with variable regions. When the TCR engages with antigenic peptide and MHC, the T lymphocyte is activated through signal transduction, that is, a series of biochemical events mediated by associated enzymes, co-receptors, specialized adaptor molecules, activated or released transcription factors.
In 1984, Tak Wah Mak and Mark M. Davis discovered the mouse TCR respectively; these findings allowed the entity and structure of the elusive TCR, known before as the "Holy Grail of Immunology", to be revealed. This allowed scientists from around the world to carry out studies on the TCR, leading to important studies in the fields of CAR-T, Cancer immunotherapy and Checkpoint inhibition; the TCR is a disulfide-linked membrane-anchored heterodimeric protein consisting of the variable alpha and beta chains expressed as part of a complex with the invariant CD3 chain molecules. T cells expressing this receptor are referred to as α:β T cells, though a minority of T cells express an alternate receptor, formed by variable gamma and delta chains, referred as γδ T cells; each chain is composed of two extracellular domains: Variable region and a Constant region, both of Immunoglobulin superfamily domain forming antiparallel β-sheets. The Constant region is proximal to the cell membrane, followed by a transmembrane region and a short cytoplasmic tail, while the Variable region binds to the peptide/MHC complex.
The variable domain of both the TCR α-chain and β-chain each have three hypervariable or complementarity determining regions. There is an additional area of hypervariability on the β-chain that does not contact antigen and, therefore, is not considered a CDR; the residues in these variable domains are located in two regions of the TCR, at the interface of the α- and β-chains and in the β-chain framework region, thought to be in proximity to the CD3 signal-transduction complex. CDR3 is the main CDR responsible for recognizing processed antigen, although CDR1 of the alpha chain has been shown to interact with the N-terminal part of the antigenic peptide, whereas CDR1 of the β-chain interacts with the C-terminal part of the peptide. CDR2 is thought to recognize the MHC. CDR4 of the β-chain is not thought to participate in antigen recognition, but has been shown to interact with superantigens; the constant domain of the TCR consists of short connecting sequences in which a cysteine residue forms disulfide bonds, which form a link between the two chains.
The TCR is a member of the immunoglobulin superfamily, a large group of proteins involved in binding and adhesion. The TCR is similar to a half-antibody consisting of a single heavy and single light chain, except the heavy chain is without its crystallisable fraction; the two subunits of TCR are twisted together. Whereas the antibody uses its Fc region to bind to Fc Receptors on leukocytes, TCR is docked onto the cell membrane. However, it is not able to mediate signal transduction itself due to its short cytoplasmic tail, so TCR still requires CD3 and zeta to carry out the signal transduction in its place, just as antibodies require binding to FcRs to initiate signal transduction. In this way the MHC-TCR-CD3 interaction for T cells is functionally similar to the antigen-immunoglobulin-FcR interaction for myeloid leukocytes, Ag-Ig-CD79 interaction for B cells; the generation of TCR diversity is similar to that for B cell antigen receptors. It arises from genetic recombination of the DNA encoded segments in individual somatic T cells by somatic VJ recombination using RAG1 and RAG2 recombinases.
Unlike immunoglobulins, however, TCR genes do not undergo somatic hypermutation, T cells do not express activation-induced cytidine deaminase. The recombination pro cess that creates diversity in BCR and TCR is unique to lymphocytes during the early stages of their development in primary lymphoid organs; each recombined TCR possess unique antigen specificity, determined by the structure of the antigen-binding site formed by the α and β chains in case of αβ T cells or γ and δ chains on case of γδ T cells. The TCR alpha chain is generated by VJ recombination, whereas the beta chain is generated by VDJ recombination. Generation of the TCR gamma chain involves VJ recombination, whereas generation of the TCR delta chain occurs by VDJ recombination; the intersection of these specific regions corresponds to the CDR3 region, important for peptide/MHC recognition. It is the un
Macrophages are a type of white blood cell, of the immune system, that engulfs and digests cellular debris, foreign substances, cancer cells, anything else that does not have the type of proteins specific to healthy body cells on its surface in a process called phagocytosis. These large phagocytes are found in all tissues, where they patrol for potential pathogens by amoeboid movement, they take various forms throughout the body. Besides phagocytosis, they play a critical role in nonspecific defense and help initiate specific defense mechanisms by recruiting other immune cells such as lymphocytes. For example, they are important as antigen presenters to T cells. In humans, dysfunctional macrophages cause severe diseases such as chronic granulomatous disease that result in frequent infections. Beyond increasing inflammation and stimulating the immune system, macrophages play an important anti-inflammatory role and can decrease immune reactions through the release of cytokines. Macrophages that encourage inflammation are called M1 macrophages, whereas those that decrease inflammation and encourage tissue repair are called M2 macrophages.
This difference is reflected in their metabolism. However, this dichotomy has been questioned as further complexity has been discovered. Human macrophages are about 21 micrometres in diameter and are produced by the differentiation of monocytes in tissues, they can be identified using flow cytometry or immunohistochemical staining by their specific expression of proteins such as CD14, CD40, CD11b, CD64, F4/80 /EMR1, lysozyme M, MAC-1/MAC-3 and CD68. Macrophages were first discovered by Élie Metchnikoff, a Russian zoologist, in 1884. A majority of macrophages are stationed at strategic points where microbial invasion or accumulation of foreign particles is to occur; these cells together as a group are known as the mononuclear phagocyte system and were known as the reticuloendothelial system. Each type of macrophage, determined by its location, has a specific name: Investigations concerning Kupffer cells are hampered because in humans, Kupffer cells are only accessible for immunohistochemical analysis from biopsies or autopsies.
From rats and mice, they are difficult to isolate, after purification, only 5 million cells can be obtained from one mouse. Macrophages can express paracrine functions within organs that are specific to the function of that organ. In the testis for example, macrophages have been shown to be able to interact with Leydig cells by secreting 25-hydroxycholesterol, an oxysterol that can be converted to testosterone by neighbouring Leydig cells. Testicular macrophages may participate in creating an immune privileged environment in the testis, in mediating infertility during inflammation of the testis. Cardiac resident macrophages participate in electrical conduction via gap junction communication with cardiac myocytes. Macrophages can be classified on basis of the fundamental activation. According to this grouping there are classically activated macrophages, wound-healing macrophages and regulatory macrophages. Macrophages that reside in adult healthy tissues either derive from circulating monocytes or are established before birth and maintained during adult life independently of monocytes.
By contrast, most of the macrophages that accumulate at diseased sites derive from circulating monocytes. When a monocyte enters damaged tissue through the endothelium of a blood vessel, a process known as leukocyte extravasation, it undergoes a series of changes to become a macrophage. Monocytes are attracted to a damaged site by chemical substances through chemotaxis, triggered by a range of stimuli including damaged cells and cytokines released by macrophages at the site. At some sites such as the testis, macrophages have been shown to populate the organ through proliferation. Unlike short-lived neutrophils, macrophages survive longer in the body, up to several months. Macrophages are professional phagocytes and are specialized in removal of dying or dead cells and cellular debris; this role is important in chronic inflammation, as the early stages of inflammation are dominated by neutrophils, which are ingested by macrophages if they come of age. The neutrophils are at first attracted to a site, where they proliferate, before they are phagocytized by the macrophages.
When at the site, the first wave of neutrophils, after the process of aging and after the first 48 hours, stimulate the appearance of the macrophages whereby these macrophages will ingest the aged neutrophils. The removal of dying cells is, to a greater extent, handled by fixed macrophages, which will stay at strategic locations such as the lungs, neural tissue, bone and connective tissue, ingesting foreign materials such as pathogens and recruiting additional macrophages if needed; when a macrophage ingests a pathogen, the pathogen becomes trapped in a phagosome, which fuses with a lysosome. Within the phagolysosome and toxic peroxides digest the pathogen. However, some bacteria, such as Mycobacterium tuberculosis, have become resistant to these methods of digestion. Typhoidal Salmonellae induce their own phagocytos
CD154 called CD40 ligand or CD40L, is a protein, expressed on activated T cells and is a member of the TNF superfamily of molecules. It binds to CD40 on antigen-presenting cells, which leads to many effects depending on the target cell type. In total CD40L has three binding partners: CD40, α5β1 integrin and αIIbβ3. CD154 acts as a costimulatory molecule and is important on a subset of T cells called T follicular helper cells. On TFH cells, CD154 promotes B cell maturation and function by engaging CD40 on the B cell surface and therefore facilitating cell-cell communication. A defect in this gene results in an inability to undergo immunoglobulin class switching and is associated with hyper IgM syndrome. Absence of CD154 stops the formation of germinal centers and therefore prohibiting antibody affinity maturation, an important process in the adaptive immune system. In 1991, three groups reported discovering CD154. Seth Lederman at Columbia University generated a murine monoclonal antibody, 5c8 that inhibited contact-dependent T cell helper function in human cells which characterized the 32 kDa surface protein transiently expressed on CD4+ T cells.
Richard Armitage at Immunex cloned a cDNA encoding CD154 by screening an expression library with CD40-Ig. Randolph Noelle at Dartmouth Medical School generated an antibody that bound a 39 kDa protein on murine T cells and inhibited helper function. Noelle contested Lederman's patent, but the challenge was rejected on all counts CD40 ligand is expressed on activated CD4+ T lymphocytes but is found in a soluble form. While CD40L was described on T lymphocytes, its expression has since been found on a wide variety of cells, including platelets, mast cells, basophils, NK cells, B lymphocytes, as well as non-haematopoietic cells. CD40L plays a central role in costimulation and regulation of the immune response via T cell priming and activation of CD40-expressing immune cells. In the macrophage, the primary signal for activation is IFN-γ from Th1 type CD4 T cells; the secondary signal is CD40L on the T cell. As a result, the macrophage expresses more CD40 and TNF receptors on its surface, which helps increase the level of activation.
The activated macrophage can destroy phagocytosed bacteria and produce more cytokines. B cells can present antigens to a specialized group of helper T cells called TFH cells. If an activated TFH cell recognizes the peptide presented by the B cell, the CD40L on the T cell binds to the B cell's CD40, causing B cell activation; the T cell produces IL-4, which directly influences B cells. As a result of this stimulation, the B cell can undergo rapid cellular division to form a germinal center where antibody isotype switching and affinity maturation occurs, as well as their differentiation to plasma cells and memory B cells; the end-result is a B cell, able to mass-produce specific antibodies against an antigenic target. Early evidence for these effects were that in CD40 or CD154 deficient mice, there is little class switching or germinal centre formation, immune responses are inhibited. Activation of endothelial cells by CD40L leads to reactive oxygen species production, as well as chemokine and cytokine production, expression of adhesion molecules such as E-selectin, ICAM-1, VCAM-1.
This inflammatory reaction in endothelial cells promotes recruitment of leukocytes to lesions and may promote atherogenesis. CD40L has shown to be a potential biomarker for atherosclerotic instability. CD154 has been shown to interact with RNF128. CD154+Antigen at the US National Library of Medicine Medical Subject Headings Human CD40LG genome location and CD40LG gene details page in the UCSC Genome Browser. GeneReviews/NCBI/NIH/UW entry on X-Linked Hyper IgM Syndrome or Immunodeficiency with Hyper-IgM, Type 1
Programmed death-ligand 1 known as cluster of differentiation 274 or B7 homolog 1 is a protein that in humans is encoded by the CD274 gene. Programmed death-ligand 1 is a 40kDa type 1 transmembrane protein, speculated to play a major role in suppressing the adaptive arm of immune system during particular events such as pregnancy, tissue allografts, autoimmune disease and other disease states such as hepatitis; the adaptive immune system reacts to antigens that are associated with immune system activation by exogenous or endogenous danger signals. In turn, clonal expansion of antigen-specific CD8 + T cells and/or; the binding of PD-L1 to the inhibitory checkpoint molecule PD-1 transmits an inhibitory signal based on interaction with phosphatases via Immunoreceptor Tyrosine-Based Switch Motif motif. This reduces the proliferation of antigen-specific T-cells in lymph nodes, while reducing apoptosis in regulatory T cells - further mediated by a lower regulation of the gene Bcl-2. PD-L1 was characterized at the Mayo Clinic as an immune regulatory molecule, B7-H1.
This molecule was renamed as PD-L1 because it was identified as a ligand of PD-1 Several human cancer cells expressed high levels of B7-H1, blockade of B7-H1 reduced the growth of tumors in the presence of immune cells. At that time it was concluded. In 2003 B7-H1 was shown to be expressed on Myeloid cells as checkpoint protein and was proposed as potential target in cancer immunotherapy in human clinic PD-L1 binds to its receptor, PD-1, found on activated T cells, B cells, myeloid cells, to modulate activation or inhibition; the affinity between PD-L1 and PD-1, as defined by the dissociation constant Kd, is 770nM. PD-L1 has an appreciable affinity for the costimulatory molecule CD80, but not CD86. CD80's affinity for PD-L1, 1.4µM, is intermediate between its affinities for CD28 and CTLA-4. The related molecule PD-L2 shares PD-1 as a receptor. Said et al. showed that PD-1, up-regulated on activated CD4 T-cells, can bind to PD-L1 expressed on monocytes and induces IL-10 production by the latter.
Engagement of PD-L1 with its receptor PD-1 on T cells delivers a signal that inhibits TCR-mediated activation of IL-2 production and T cell proliferation. The mechanism involves inhibition of ZAP70 phosphorylation and its association with CD3ζ. PD-1 signaling attenuates PKC-θ activation loop phosphorylation, necessary for the activation of transcription factors NF-κB and AP-1, for production of IL-2. PD-L1 binding to PD-1 contributes to ligand-induced TCR down-modulation during antigen presentation to naive T cells, by inducing the up-regulation of the E3 ubiquitin ligase CBL-b. Upon IFN-γ stimulation, PD-L1 is expressed on T cells, NK cells, myeloid DCs, B cells, epithelial cells, vascular endothelial cells; the PD-L1 gene promoter region has a response element to the interferon regulatory factor. Type I interferons can upregulate PD-L1 on murine hepatocytes, monocytes, DCs, tumor cells. PD-L1 is notably expressed on macrophages. In the mouse, it has been shown that classically activated macrophages upregulate PD-L1.
Alternatively, macrophages activated by IL-4 upregulate PD-L1, while upregulating PD-L2. It has been shown by STAT1-deficient knock-out mice that STAT1 is responsible for upregulation of PD-L1 on macrophages by LPS or interferon-gamma, but is not at all responsible for its constitutive expression before activation in these mice. Resting human cholangiocytes express PD-L1 mRNA, but not the protein, due to translational suppression by microRNA miR-513. Upon treatment with interferon-gamma, miR-513 was down-regulated, thereby lifting suppression of PD-L1 protein. In this way, interferon-gamma can induce PD-L1 protein expression by inhibiting gene-mediated suppression of mRNA translation. PD-L1 promoter DNA methylation may predict survival in some cancers after surgery, it appears. An analysis of 196 tumor specimens from patients with renal cell carcinoma found that high tumor expression of PD-L1 was associated with increased tumor aggressiveness and a 4.5-fold increased risk of death. Many PD-L1 inhibitors are in development as immuno-oncology therapies and are showing good results in clinical trials.
Clinically available examples include Durvalumab and avelumab. In normal tissue, feedback between transcription factors like STAT3 and NF-κB restricts the immune response to protect host tissue and limit inflammation. In cancer, loss of feedback restriction between transcription factors can lead to increased local PD-L1 expression, which could limit the effectiveness of systemic treatment with agents targeting PD-L1. In a mouse model of intracellular infection, L. monocytogenes induced PD-L1 protein expression in T cells, NK cells, macrophages. PD-L1 blockade resulted in increased mortality for infected mice. Blockade reduced TNFα and nitric oxide production by macrophages, reduced granzyme B production by NK cells, decreased proliferation of L. monocytogenes antigen-specific CD8 T cells. This evidence suggests that PD-L1 acts as a positive costimulatory molecule in intracellular infection; the PD-1/PD-L1 interaction is implicated in autoimmunity from several lines of evidence. NOD mice, an animal model for autoimmunity tha
In molecular biology, CD4 is a glycoprotein found on the surface of immune cells such as T helper cells, monocytes and dendritic cells. It was discovered in the late 1970s and was known as leu-3 and T4 before being named CD4 in 1984. In humans, the CD4 protein is encoded by the CD4 gene. CD4 + T helper cells are white blood cells, they are referred to as CD4 cells, T-helper cells or T4 cells. They are called helper cells because one of their main roles is to send signals to other types of immune cells, including CD8 killer cells, which destroy the infectious particle. If CD4 cells become depleted, for example in untreated HIV infection, or following immune suppression prior to a transplant, the body is left vulnerable to a wide range of infections that it would otherwise have been able to fight. Like many cell surface receptors/markers, CD4 is a member of the immunoglobulin superfamily, it has four immunoglobulin domains that are exposed on the extracellular surface of the cell: D1 and D3 resemble immunoglobulin variable domains.
D2 and D4 resemble immunoglobulin constant domains. The immunoglobulin variable domain of D1 adopts an immunoglobulin-like β-sandwich fold with seven β-strands in 2 β-sheets, in a Greek key topology. CD4 interacts with the β2-domain of MHC class II molecules through its D1 domain. T cells displaying CD4 molecules on their surface, are specific for antigens presented by MHC II and not by MHC class I. MHC class I contains Beta-2 microglobulin; the short cytoplasmic/intracellular tail of CD4 contains a special sequence of amino acids that allow it to recruit and interact with the tyrosine kinase Lck. CD4 is a co-receptor of the T cell receptor and assists the latter in communicating with antigen-presenting cells; the TCR complex and CD4 each bind to distinct regions of the antigen-presenting MHCII molecule - α1/β1 and β2, respectively. In CD4 the interaction involves its extracellular D1 domain; the resulting close proximity between the TCR complex and CD4 allows the tyrosine kinase Lck bound to the cytoplasmic tail of CD4 to tyrosine-phosphorylate the Immunoreceptor tyrosine activation motifs on the cytoplasmic domains of CD3 to amplify the signal generated by the TCR.
Lck is essential for the activation of many molecular components of the signaling cascade of an activated T cell. Depending on the signal, different types of T helper cells result. Phosphorylated ITAM motifs on CD3 recruit and activate SH2 domain-containing protein tyrosine kinases such as Zap70 to further mediate downstream signalling through tyrosine phosphorylation, leading to transcription factor activation including NF-κB and consequent T cell activation. CD4 has been shown to interact with SPG21, Lck and Protein unc-119 homolog. HIV-1 uses CD4 to gain entry into host T-cells and achieves this through its viral envelope protein known as gp120; the binding to CD4 creates a shift in the conformation of gp120 allowing HIV-1 to bind to a co-receptor expressed on the host cell. These co-receptors are chemokine receptors CCR5 or CXCR4. Following a structural change in another viral protein, HIV inserts a fusion peptide into the host cell that allows the outer membrane of the virus to fuse with the cell membrane.
HIV infection leads to a progressive reduction in the number of T cells expressing CD4. Medical professionals refer to the CD4 count to decide when to begin treatment during HIV infection, although recent medical guidelines have changed to recommend treatment at all CD4 counts as soon as HIV is diagnosed. A CD4 count measures the number of T cells expressing CD4. While CD4 counts are not a direct HIV test—e.g. They do not check the presence of viral DNA, or specific antibodies against HIV—they are used to assess the immune system of a patient. National Institutes of Health guidelines recommend treatment of any HIV-positive individuals, regardless of CD4 count Normal blood values are expressed as the number of cells per microliter of blood, with normal values for CD4 cells being 500–1200 cells/mm3. Patients undergo treatments when the CD4 counts reach a level of 350 cells per microliter in Europe but around 500/μL in the US. Medical professionals refer to CD4 tests to determine efficacy of treatment.
Viral load testing provides more information about the efficacy for therapy than CD4 counts. For the first 2 years of HIV therapy, CD4 counts may be done every 3–6 months. If a patient's viral load becomes undetectable after 2 years CD4 counts might not be needed if they are above 500/mm3. If the count remains at 300–500/mm3 the tests can be done annually, it is not necessary to schedule CD4 counts with viral load tests and the two should be done independently when each is indicated. CD4 continues to be expressed in most neoplasms derived from T helper cells, it is therefore possible to use CD4 immunohistochemistry on tissue biopsy samples to identify most forms of peripheral T cell lymphoma and related malignant conditions. The antigen has been associated with a number of autoimmune diseases such as vitiligo and type I diabetes mellitus. T-cells play a large part in autoinflammatory diseases; when testing a drug's efficacy or studying diseases, it is helpful to quantify the amount of T-cells. on fresh-frozen tissue with CD4+, CD8+, CD3+ T-cell markers.
CD4+ T cells and antitumor immunity CD1+Ant
A knockout mouse or knock-out mouse is a genetically modified mouse in which researchers have inactivated, or "knocked out", an existing gene by replacing it or disrupting it with an artificial piece of DNA. They are important animal models for studying the role of genes which have been sequenced but whose functions have not been determined. By causing a specific gene to be inactive in the mouse, observing any differences from normal behaviour or physiology, researchers can infer its probable function. Mice are the laboratory animal species most related to the humans for which the knockout technique can be applied, they are used in knockout experiments those investigating genetic questions that relate to human physiology. Gene knockout in rats is much harder and has only been possible since 2003; the first recorded knockout mouse was created by Mario R. Capecchi, Martin Evans, Oliver Smithies in 1989, for which they were awarded the 2007 Nobel Prize in Physiology or Medicine. Aspects of the technology for generating knockout mice, the mice themselves have been patented in many countries by private companies.
Knocking out the activity of a gene provides information about what that gene does. Humans share many genes with mice. Observing the characteristics of knockout mice gives researchers information that can be used to better understand how a similar gene may cause or contribute to disease in humans. Examples of research in which knockout mice have been useful include studying and modeling different kinds of cancer, heart disease, arthritis, substance abuse, anxiety and Parkinson's disease. Knockout mice offer a biological and scientific context in which drugs and other therapies can be developed and tested. Millions of knockout mice are used in experiments each year. There are several thousand different strains of knockout mice. Many mouse models are named after the gene, inactivated. For example, the p53 knockout mouse is named after the p53 gene which codes for a protein that suppresses the growth of tumours by arresting cell division and/or inducing apoptosis. Humans born with mutations that deactivate the p53 gene suffer from Li-Fraumeni syndrome, a condition that increases the risk of developing bone cancers, breast cancer and blood cancers at an early age.
Other mouse models are named according to their physical behaviours. There are several variations to the procedure of producing knockout mice; the gene to be knocked out is isolated from a mouse gene library. A new DNA sequence is engineered, similar to the original gene and its immediate neighbour sequence, except that it is changed sufficiently to make the gene inoperable; the new sequence is given a marker gene, a gene that normal mice don't have and that confers resistance to a certain toxic agent or that produces an observable change. In addition, a second gene, such as herpes tk+, is included in the construct in order to accomplish a complete selection. Embryonic stem cells are grown in vitro. For this example, we will take stem cells from a white mouse; the new sequence from step 1 is introduced into the stem cells from step 2 by electroporation. By the natural process of homologous recombination some of the electroporated stem cells will incorporate the new sequence with the knocked-out gene into their chromosomes in place of the original gene.
The chances of a successful recombination event are low, so the majority of altered cells will have the new sequence in only one of the two relevant chromosomes – they are said to be heterozygous. Cells that were transformed with a vector containing the neomycin resistance gene and the herpes tk+ gene are grown in a solution containing neomycin and Ganciclovir in order to select for the transformations that occurred via homologous recombination. Any insertion of DNA that occurred via random insertion will die because they test positive for both the neomycin resistance gene and the herpes tk+ gene, whose gene product reacts with Ganciclovir to produce a deadly toxin. Moreover, cells that do not integrate any of the genetic material test negative for both genes and therefore die as a result of poisoning with neomycin; the embryonic stem cells that incorporated the knocked-out gene are isolated from the unaltered cells using the marker gene from step 1. For example, the unaltered cells can be killed using a toxic agent to which the altered cells are resistant.
The knocked-out embryonic stem cells from step 4 are inserted into a mouse blastocyst. For this example, we use blastocysts from a grey mouse; the blastocysts now contain two types of stem cells: the original ones, the knocked-out cells. These blastocysts are implanted into the uterus of female mice, where they develop; the newborn mice will therefore be chimeras: some parts of their bodies result from the original stem cells, other parts from the knocked-out stem cells. Their fur will show patches of white and grey, with white patches derived from the knocked-out stem cells and grey patches from the recipient blastocyst; some of the newborn chimera mice will have gonads derived from knocked-out stem cells, will therefore produce eggs or sperm containing the knocked-out gene. When these chimera mice are crossbred with others of the wild type, some of their offspring will have one copy of the knocked-out gene in all their cells; these mice will be white and are not chimeras, however they are still heterozygous.
When these heterozygous offspring are interbred, some of their offspring will inherit