Assignment of Immunology

Department of Bio-Informatics

3 rd Semester (Morning)

Subject Immunology

Teacher Sir Asad Aslam

Roll No. 1255, 1259, 1262, 1274

Topic Nude Mice

G.C. University Faisalabad

Topic NUDE MOUSE

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Assignment of Immunology

Knockout Mouse

A knockout mouse is a genetically engineered mouse in which one or more genes have been turned off through a gene knockout. Knockout mice are important animal models for studying the role of genes which have been sequenced, but have unknown functions. By causing a specific gene to be inactive in the mouse, and observing any differences from normal behavior or condition, researchers can infer its probable function.

The first knockout mouse was created by Mario R. Capecchi, Martin Evans and Oliver Smithies in 1989, for which they were awarded the Nobel Prize for Medicine in 2007.

How to make knockout mouseThere are several techniques to make knockout mouse:Gene Knockout by Targeted Disruption:

The technique of gene targeting takes advantage of the phenomenon known as homologous recombination.  Cloned copies of the target gene are altered to make them nonfunctional and are then introduced into the ES cell where they recombine with the homologous gene in the cell’s genome, replacing the normal gene with a nonfunctional copy.

A second powerful technique achieves tissue-specific or developmentally regulated gene deletion by employing the DNA sequences and enzymes used by bacteriophage P1 to excise itself from a host cell’s genome.

Inducible Knockout Mouse:

Knock outs which are embryonic lethals or toxic can be made inducible. This has the advantage of allowing the knockout to take place AFTER development/ embryogenesis has occurred. This is accomplished by using a Tet On / Tet Off system.

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THE NUDE MOUSE

ude mouse is a genetic mutant that has a deteriorated or removed thymus gland, resulting in an inhibited immune system due to a greatly reduced number of T cells.

The phenotype or main outward appearance of the mouse is a lack of body hair, which gives it the "nude" nickname. The first nude mutation was observed in 1962. The Nude mouse is valuable to research because it can receive many different types of tissue and tumor grafts, as it mounts no rejection response. These xenografts are commonly used in research to test new methods of imaging and treating tumors. The genetic basis of the nude mouse mutation is a disruption of a gene named the FOXN1 gene.

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~~History and Nomenclature~~

Nude mice have been bred since at least 1937. They were discovered and first bred by Miroslav Holub (1923-1998), a distinguished Czech immunologist and a renowned poet.

The Nomenclature for the nude mouse has changed several times since their discovery. Originally they were described as ‘nu’ and this was updated to ‘Hfh11nu’ when the mutated gene was identified as a mutation in the HNF-3/forkhead homolog 11 gene. Then in 2000 the gene responsible for the mutation was identified as a member of the Fox gene family and the nomenclature was updated to ‘Foxn1nu’.

~~Significance~~

A mutant mouse said to be nude because it is hairless due to the presence of two copies of the gene ‘nu’. Because they lack a thymus, Nude mice cannot generate mature T lymphocytes. Therefore they are unable to mount most types of immune responses, including:

1. antibody formation that requires CD4+ helper T cells2. cell-mediated immune responses, which require CD4+ and/or CD8+ T cells3. delayed-type hypersensitivity responses (require CD4+ T cells)4. killing of virus-infected or malignant cells (requires CD8+ cytotoxic T cells)5. graft rejection (requires both CD4+ and CD8+ T cells)

Because of the above features, nude mice have served in the laboratory to gain insights into the immune system, leukemia, solid tumors, AIDS and other forms of immune deficiency as well as leprosy. Although, the Nude mouse lacks T cells, it has a normal complement of bone marrow-dependent B cells. Nude mice also have elevated levels of both macrophages and NK cells, their macrophages are also more potent than those from mice with a normal functioning thymus. Moreover, the absence of functioning T cells prevents Nude mice from rejecting not only allografts, but they cannot even reject xenografts.

Some other characteristics include: A high incidence of glomerulonephritis by three months of age. This includes higher

than normal deposits of IgG, IgM, and IgA. Have normal hair growth cycles despite a hairless state Have a low incidence of nonsuppurative dermatitis Low incidence of keratoacanthoma and squamous carcinoma Very high incidence of corneal vascularization

“Most strains of nude mice are slightly ‘leaky’ and do have a few T cells, especially as they age. For this reason, nude mice are less popular in research today, since knockout mice with more complete defects in the immune system have been constructed e.g. RAG1 and RAG2 knockout mice.”

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~~Genetics~~

The basic Genetics of Nude mice is that Nude mice have a spontaneous deletion in the FOXN1 gene. Humans with mutations in FOXN1 also are athymic and immune deficient. Mice with a targeted deletion in the FOXN1, knockout mice, also show the "nude" phenotype. Mice homozygous for the nu mutation have a basic defect that is thought to involve all three embryonic germ layers. The thymic dysgenesis appears to be caused by an ectodermal and endodermal metabolic error and inadequate mesenchymal mass in the mediastinal area. This abnormality results in a failure in the homing of prothymocytes to the developing thymic rudiment, accompanied by a deficiency of Ia+

stromal cells and hyperplasia of muco-polysaccharide producing epithelial cells, which form peculiar structures. In the skin, there is defective keratinization of hair and degeneration of hair follicles, resulting in hairlessness despite an increased frequency of hair growth cycles.

Since nude females have under-developed mammary glands and are unable to effectively nurse their young, nude males are bred with heterozygous females. Athymic Nude mice are produced by breeding heterozygous females with homozygous males. While homozygous females will carry litters to term, most pups will not survive due to poor lactation.

Picture: Placental and foetal weight and litter size were studied on the 18th day of pregnancy in the athymic nude mouse. Placental weight and litter size were unaffected by the absence of a thymus. Foetal weight was reduced, probably as a result of the health status of such animals.

~~Immunological Responses of Nude Mice~~

Mice homozygous for the mutation nude lack a thymus. They are deficient in lymphocytes in thymus dependent areas of peripheral lymphoid organs. They lack cells bearing ‘θ’ which is a cell surface antigen found on thymocytes and thymus dependent cells. In a preliminary communication it have been reported that nu/nu mice have impaired heterograft rejection and low immunoglobulin levels.

In 1969 Miller and Mitchell gave evidences, that thymus-derived cells and antibody producing cells form two populations, but nu/nu mice shows a deficiency in both thymus-derived cells and in antibody forming cells, but how both the populations are effected? This was defined as, it could occur either because the nude mutation affects the

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Assignment of Immunology

maturation/differentiation of both cell types, for instance, by preventing differentiation of a common precursor, or may be because the normal development of one cell type is dependent on the normal development of the other.

There is ample evidence that in the absence of a thymus-derived cell population there is retarded development of the antibody forming cell population. The observed fact that nu/nu spleen cells will synergize with thymocytes, suggests that, nude mice do not lack precursors of antibody forming cells, but that they remain precursors in the absence of a thymus-derived cell population. Therefore, the low Plaque Forming Cells (PFC) response and the low immunoglobulin levels of Nude mice are a secondary effect of an absent thymus-derived cell population.

Nevertheless, Nude mice do have some immunoglobulin and do produce some PFC in response to sheep RBC. This kind of act suggests either that there are some mature thymus-derived cells in Nude mice that came from their mothers or that antibody forming cells can mature in the absence of a thymus at a slow rate. At the moment it is not possible to rule out either possibility.

The low Immunoglobulin level is suggestive to be as due to B-cell defect. If Nude mice do lack a normal B cell population, the interesting question arises as to the primary site of the block. nudes lack mature T cells, but there is no intrinsic defect in T cell precursors, rather it is the lack of the normal inductive environment provided by the thymus that causes the block in T cell differentiation. The proposed block in B cell development imposed by nude could either be intrinsic to B cell precursors or secondary to a defect in a tissue that normally induces B cell differentiation. If a defect in inductive tissue exists, it is an entirely open question as to whether the thymus itself provides the inductive signals, for instance, by means of a hormone.

~~What if Thymus is grafted? ~~

The peripheral lymphoid organs in the Nude mice are characterized by two major abnormalities: (1) a striking lymphocyte depletion of the lymph node and Payer’s patches thymus-dependent areas which are accompanied by poor post-capillary venule definition and (2) a virtual absence of germinal centres.

It was suggested that this apparent failure of germinal centre formation might be the indirect consequence of the absence of thymus-derived cells in the Nude mice. In view of the differences between the response of nude and of thymectomized mice, it was confirmed that well-defined germinal centres had clearly developed at 7 days after antigen, ‘Pneumococcus polysaccharide’, stimulation in C3H/Bi mice, which were thymectomized within 48 and 72 hours of birth. In addition, it was also observed that germinal centres were present in the nu/nu thymus graft recipients not deliberately submitted to antigen stimulation.

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The mechanism by which a few thymus-derived cells might influence germinal centre development is not clear. A number of possibilities should be considered:(a) thymus-derived cells are themselves integral components of germinal centres(b) thymus-derived cells penetrate the primary nodules and release factors capable of inducing non thymus-derived cells to transform and divide(c) germinal centre development depends on the presence of normal levels of certain immunoglobulin subclasses whose synthesis in turn is dependent on the specific interaction between T and B cells and(d) germinal centre cells are exquisitively sensitive to abnormal hormonal levels and their absence may be associated with subtle endocrinological deficiencies

The possibility that thymus-derived cells release factors capable of inducing neighboring cells to transform and divide, seems plausible indeed, based on the finding of lower number of mitotic figures in the germinal centre of thymectomized mice than in those of intact controls, and before it became apparent that the thymus exports large numbers of cells into circulation, it was postulated that the profound lymphopenia and lymphocyte depletion of the peripheral lymphoid organs of thymectomized mice were due to lack of mitotic stimulation of germinal centre cells.

Another factor that may be of importance in normal germinal centre development is the class of immunoglobulin produced in response to antigenic stimulation. Nude mice have a deficiency in serum λA, λG2a and λG1 which is corrected by thymus grafting or the intra-peritoneal injection of thymus cells, therefore the absence of germinal centres in these animals may be the reflection of the low concentration of one or other of these immunoglobulins. An alterative that cannot be excluded, to explain the presence of germinal centres in the nude thymus graft recipients is that they are the reflection of some form of specific T/B interaction in response to some unidentified antigenic stimulation.

The infilling of the thymus-dependent zones with lymphocytes, after thymus grafting varied not only between different animals, but also between different organs within the same animal. Nevertheless, the overall length of time of this process was similar, though perhaps a little slower than the repopulation time of neonatally thymectomized mice following implantation of a syngeneic thymus graft under the kidney capsule. It also increases the Interferon levels that were low due to lack of Thymus.

Two major questions remain to be answered.

Is the absence of a thymus-derived cell population due to an abnormal development of the thymus epithelium or of the thymocytes precursor? A simplified view of the development

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of the antibody forming system is presented in figure. Four separate cell lines are involved. Three cell-cell or tissue-tissue interactions are known. The interaction of thymus-derived cells and antibody forming precursors has been referred to above. This has been demonstrated that the mature thymocytes are the product of an interaction of thymic epithelium with a stem cell. Slightly more conjectural is the interaction of mesenchyme and thymic epithelium afilage. Just where in this picture the nude mutation has its first effect is still unknown.

This raises the final question. Can the pleiotropic effects of nude be explained by a single defect, e.g. abnormal development of epithelial tissue? It is tempting to make this speculation, but nude has yet to be shown to be a mutation involving a single locus.

~~Uses of Nude Mouse~~

“Not only have Nude mice answered many questions in immunology, but their ability to maintain human tissues have made them useful tools for investigating, for example, the properties of human cancers.”

Many of the uses of Nude mice are:

Self-recognition specificity expressed by T cells from nude mice, Absence of detectable Ia-restricted T cells in nude mice that do Exhibit self-K/D-restricted T cell responses

Functionality of T cells in athymic nude mice if inserted from mice having thymus Lack of mature B cells in nude mice With X-linked immune deficiency which shows

that lack of B-cells in nu/nu mice is due to a recessive gene associated with nu/nu mice and X-linked immune deficiency

It is also observed fact that truncated Galectin-3 inhibits tumor growth and metastasis in Orthotopic Nude mice model of human Breast cancer

Suppression of Tumor cell growth was observed both in Nude Mice and in culture by n-3 Polyunsaturated Fatty acids when mediated through Cyclooxygenase-independent pathways

The influence of synthetic polymeric agents on the immune responsiveness of congenitally athymic (nude) mice was investigated by determining the effects of in vivo treatment with polynucleotides and polymeric haptenated antigens on splenic theta-bearing cells, on mitogen stimulation and on plaque-forming cell responses to thymic dependent and thymic independent antigens. Contrary to in vitro data, no evidence was obtained to demonstrate in vivo restoration of these immune parameters by the use of non-specific immune enhancers

Nude mice are now a days widely used by combining two different deficient strains, as Nude mice and xid mice, Nude mice and SCID mice etc

The H-2 complex and M locus have been defined for two incipient inbred strains of mice carrying the nu mutation. The strains differ at both loci from each other and cells from nu/nu mice of these allogeneic strains have been used to examine the role of T cells in the one-way mixed leukocyte reaction (MLR)

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Antibody response to many of tumor causing viruses, for example, Simian virus tumor antigen when reconstituted with T-cells

These six photographs show 6 different skin xenografts surviving on nude mice.

A. Human (mammal) skin after 60 days.

B. Cat (mammal) skin at 51 days.

C. Chicken (bird) at 32 days; the feathers were already present when the graft was made.

D. Chameleon (reptile) at 41 days.

E. Fence lizard (reptile) at 28 days.

F. Tree frog (amphibian) at 40 days.

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~~References~~

The Nude Mouse in Experimental and Clinical Research (Vol.1). Fogh, J; Giovanella, B.C. (eds) Academic Press, 1978, ISBN 0-12-261860-2

The Nude Mouse in Experimental and Clinical Research (Vol.2). Fogh, J; Giovanella, B.C. (eds) Academic Press, 1982, ISBN 0-12-261862-9

Handbook of Mouse Mutations with Skin and Hair Abnormalities: Animal Models and Biomedical Tools By John P. Sundberg Published by CRC Press, 1994

http://jaxmice.jax.org/faq/bulletin/bulletin06.html

http://jem.rupress.org

http://www.karger.com

(OMIM)http://en.wikipedia.org/wiki/Mendelian_Inheritance_in_Man

Benjamin Cohen and David Ng, Artwork by Jane Wang – 2006

Proc. Natl. Acad. Sci. USA Vol. 81, pp. 886-888, February 1984 Medical Sciences

Proc. Natl. Acad. Sci. USA Vol. 81, pp. 886-888, February 1984 Medical Sciences

Clin. exp. Immunol. (1971) 8, 305-317 by H. H. Wortis

Immunology, 1974, 26, 769 by Maria De Sousa And Helen Pritchard

J. gen. ViroL (I976), 32, I49-152

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