Severe Combined Immunodeficient Mice By Nasarullah Khan

Introduction:
SCID was rst recognized as a rare primary immunodeciency of humans that is defined by a congenital absence of functional B and T cells. In 1983 , Dr. M.J Bosma of the Fox Chase Cancer Centre in Philadelphia, Pennsylvania, first described a mouse showing SCID phenotype. These mice show severe lymphopenia and agammaglobulinemia and are susceptible to a variety of infections. Only when SCID mice are isolated in specic pathogen-free barriers can they be maintained and kept from succumbing to fatal infections. The propagation of these mice has led to their use as an experimental tool in studies of normal immune system components as well as various host-pathogen interactions.

Genetic Basis of SCID Mice:

SCID mice possess a genetic autosomal recessive mutation designated as Prkdcscid , discovered in a BALB/c Ighb (C.B 17 Icr) mice a strain congenic to the BALB/c carrying the Igh- 1b (heavy chain immunoglobulin )allele from C57BL/Ka strain. The mutation is due to a single nucleotide substitution in the Prkdc gene located on chromosome number 16.These mice are homozygous for the Prkdc scid allele that is situated at the centromeric end of chromosome 16.Prkdc encodes for a catalytic subunit of the DNA activated protein kinase. This enzyme is involved in the annealing of the DNA ends, that is cut and rearranged (coding joint formation) as a part of the process of producing antigen specific receptors. . The role that the kinase plays in the repair of double-stranded DNA breaks remains to be claried; possibilities include (i) an initial signal to the DNA damage repair machinery on DNA damage and (ii) alteration of the chromatin structure around a DNA lesion, allowing for access of the DNA repair machinery. It is also involved in recombining the variable (V), diversity (D), and joining (J) segments of immunoglobulin and T cell receptor genes. The SCID mutation results in premature stop codon that prevents the translation of 83 amino acids at the C terminus of a protein, therefore substantially reducing the DNA- PK activity. Both B and T cells utilize a gene rearrangement strategy to generate high levels of receptor diversity without using a large percentage of the germ line . In this process, the variable(V), diversity (D), and joining (J) regions targeted for rearrangement are anked by conserved signal sequences. Protein-protein interactions involving these signal sequences bring the targeted areas

of the DNA into close proximity, and blunt-end double-stranded breaks are then formed in a sitespecic manner. A re-sorting of the ensuing four free DNA ends occurs, and the formation of two new junctions results in proper rearrangement and coding for a functional receptor. Failure of proper joining of these ends results in the failure to produce the antigen-specic receptors of the B and T cells. As the surface expression of these receptors is an important checkpoint in the development of these cells, the cells fail to mature beyond this initial stage. Therefore as SCID mice cannot complete V(D)J gene recombination, their T and B lymphocyte progenitor cells do not mature and therefore cannot develop cell mediated and humoral adaptive immune responses. The myeloid cell lineage is not affected by this mutation so there is a normal activity of granulocytes, macrophages and NK cells. Since the scid gene is 1 of 15 genes that have been implicated in this rearrangement process. A clue to the function that the scid gene product may have in this process is that SCID mice not only have defective T and B cells but also show an inability to repair double-stranded DNA breaks after exposure to irradiation or chemical agents . The common link between these two defects is the recombination of the four single-stranded DNA ends into properly joined DNA segments. Common Characteristics of SCID mice: Some of the common features of SCID mice are outlined as follows: y The immunodeficiency in mice is manifest in a number of characteristics including lymphopenia, agammaglobulinemia and a very high susceptibility to infection with viruses, bacteria and other microorganisms. When housed under conventional conditions it is not uncommon for these animals to develop life threatening infections due to Pneumocystis.murina, adventitious rodent viruses and bacteria. They have lymph nodes and thymus that are abnormally small but present. The thymus has a rudimentary medulla without a cortex while the spleen and lymphnodes have follicles that are devoid of lymphocytes. SCID mice are deficient in epidermal and follicular dendritic Thy 1 cells. This deficiency leads to an inability to produce antibodies to common antigens and an inability to reject allogenic grafts. The spleen cells do not proliferate in response to T and B cell specific mitogens. About 15% of the SCID mice spontaneously develop thymic lymphomas. The percentage of leaky SCID mice amongst young adults varies from 2-10%.

y y

Purposes of SCID Mice:

SCID mice are routinely used as model organisms for research into the basic biology of the immune system (innate and adaptive immunity), autoimmunity ,hematopoiesis, cancer ,cell transplantation strategies, the analyse effects regenerative medicine in vivo and the effects of disease on mammalian systems. They have been extensively used as hosts for normal and malignant tissue transplants. In addition, they are useful for testing the safety of new vaccines or therapeutic agents in immune compromised individuals. A great majority are also used as recipients of xenogenic and allogenic tissue grafts . As a result the desirable criteria for SCID mice are the acceptance and growth of xenografts, consistent performance across groups over time and the easy evaluation of implanted xenografts. They accept xenografts more easily as compared to the athymic nude mice due to their more profound immune dysfunction characterized by their inability to fight infections, and to reject tumors and transplants. Some scid mice have such a superior ability to engraft and support the development of human tissues and microenvironments (e.g., primary human tumors with intact stroma, a functional human immune system), they can be "humanized.".In addition SCID mice are ideal for the growth of hybridoma in vivo to produce a continuous supply of antibody (Ab). Sometimes referred to as a reagent, Ab is necessary for a wide range of diagnostic, clinical and experimental procedures. Recently SCID mice are also being used for the study of host response to parasitic diseases. We can dene three general ways in which SCID mice have been used to study parasitic diseases . First, for several parasitic diseases for which a murine or small-animal model was lacking, infection has been successfully established in SCID mice. In such instances, the successful infection of the SCID mouse has served to greatly advance the ease with which the parasitic disease can be studied. Second, establishment of a parasitic infection in SCID mice provides the opportunity to examine the role of individual components of the specic immune response in disease. Because SCID mice lack B and T cells, they can be reconstituted with dened populations of naive or immune lymphocytes which will all be of donor origin. The elucidation of pathways of innate immunity in SCID mice has been made possible by exposing them to different pathogens causing initiation of cytokine cascades involving only the macrophages, natural killer cells and neutrophils. These cascade is capable of activating these cells to heightened levels of phagocytic and bactericidal activity .Therefore the individual role of the three major cells of the innate immune system has also been clarified. By transferring the scid mutation (either alone or with other mutations and/or transgenes) to various backgrounds, a variety of scid mice has been produced, each with unique engraftment properties and research applications. For example, some are more tolerant of radiation and live longer, some are not leaky, some are better suited for vaccine development, some are better suited for HIV/AIDS research, and some can be better humanized. Since SCID mice readily support normal lymphocyte differentiation and can be reconstituted with normal lymphocytes from other mice and even partially reconstituted with human lymphocytes and this way can be used to study the effect of different diseases of humans.

SCID mice when used in cancer research can be used to study the initiation and growth of different tumors. These tumors can likewise be exposed to radiotherapy and chemotherapy in order to analyse their effect in reducing their growth. Hence , this information helps the researchers to develop possible cures and treatments of cancer. Leaky SCID Mice: One potential problem in using the SCID mouse as an immunologic tool is that the SCID defect is not absolute. Some animals are capable of carrying out functional V(D)J and VJ rearrangements and generating functional T and B cells . These animals are termed leaky; they can be detected by monitoring SCID mice for the presence of immunoglobulin G in serum. There is no standard criteria for leakiness threshold , the age at which leakiness is determined or what immunoglobulin classes should be measured. The leakiness determination varies by measured Ig class (IgM, IgG or total Ig) and the threshold for calling an animal leaky varies with the producer and researcher. Dr Melvin Bosma the discoverer of SCID mice placed the leaky threshold at less than 50g/ml of all classes of Ig measured at 100-200 days of age. Some producers only measure IgG suggesting it to be more indicative of mature antibody response, whereas IgM produced early in the antibody response may be less indicative of the specific antigen response. In immunocompetent mice in vivo IgM averages approximately 1000g/ml or 5% of the 20000g/ml total serum immunoglobulin. In contrast the total level of serum immunoglobulin in leaky mice is 50-100g/ml or less than 1% of that in immunocompetent mice .In addition the Ig produced is mostly monoclonal and oligoclonal as compared to polyclonal as would be in an immunocompetent mouse. The lack of a broad polyclonal production of immunoglobulins in leaky mice suggests that immunoglobulin is not targeted against specific antigens, but rather results from an uncontrolled production of a few B cell clones. As many B cells cloned from leaky mice have shown identical V(D)J rearrangements and identical specicity. In addition the small amount of immunoglobulin raises questions as to its biological significance especially regarding suppression of xenograft growth a function in which cell mediated immunity is considered to be more important than humoral immunity. The molecular basis of leakiness in SCID mice is not well characterized. The two possibilities being considered for the induction of leakiness in SCID mice are genetic and environmental. Dr.Bosma claims that there is no genetic basis. When he carried out selective breeding of original SCID mice he did not show any effects of lineage. However other SCID mouse producers claim that when the SCID mutation is placed on different background strains there are different leakiness rates that tend to support the genetic basis. Environmental differences seem to be more responsible for leakiness. As a higher level of stimulus of the hosts defense system, such as through pattern recognition receptors, would result in greater attempted activation of the immune system. Leaky phenotype increases with age and antigen exposure and can be minimized by housing animals in as clean an environment as possible and by using young SCID mice. By 10-14 months of age, virtually all SCID mice are leaky.

Leakiness does not have a significant effect on xenograft studies as has been evidenced according to a study carried out by Dr.Bosma on the CB-17 leaky SCID mice. The study has shown that these mice neither show a response to the mitogens, nor they have L y5+ cells in the spleen indicating that the animals were not able to respond properly to the immune stimuli. In the same study Dr. Bosma found that these mice had a higher rejection rate for allografts. Transportation Measures For SCID mice: Due to the SCID mouses severe combined immunodeficiency, their exposure to potentially pathogenic organisms must be prevented. The SCID mice are shifted from their isolator colonies into the sterile laminar flow work stations for the purpose of transportation . From those work stations these mice are packed into small isolator like containers that have been presterlized. These shipping containers i.e (Gnotosafe shippers) are preferably used for shipping SCID mice. Other light weight shipping devices that resemble flexible film isolators can also be used for a direct transfer to the isolator production colonies. Other plastic transit cages can also be used for the transportation of these mice with a plastic sleeve that can be connected to an isolator for transferring the mice without any contamination while handling. The cage and lid are constructed of molded polypropylene #5 plastic for high durability and resistance to gnawing by mice. These cages are capable of protecting its contents from contamination by murine viruses. The outside dimensions of the cages are 22" (long) by 16" (wide) by 7" (deep).Sloped sides, stacking mounts and air vents on the sides and lid allow sufficient air flow around the cage for proper ventilation. The filter material is a spunbonded polypropylene media which provides optimum strength; the continuous filaments in the media prevent tearing and slipping. All materials are autoclavable and washable in high temperature cage washers. They can be easily opened by lifting the lid upward from the front handle. Since there are no staples and wire to contend with it eliminates cuts and scrapes to the handlers. Security ties ensure that the cage is not opened during transit. For international shipments, an optional clear lid is available as an alternative to the standard opaque lid. Aseptic techniques should be used in transferring the animals from transit cages to filter-top cages. Before the transit cages are opened, they should be sprayed with a sterilant. After spraying, the cages should be placed in a laminar air flow hood, opened by personnel wearing gloves or with instruments wet with sterilant, and the animals transferred to sterilized filter-top cages. Below mentioned are the number of mice of different ages that can be shipped using Taconic transit cages: Age Range Number of Mice/TTC 25

3-6 weeks of age

7 or more weeks of age Female with Litter Timed Pregnants Retired Breeders 4 20 25

25

Taconic germ free shippers are also used for the transport of SCID mice . The germfree shipper preserves the germfree health status of the animals during shipping . The germfree shipper is a large, flexible vinyl sleeve with filtered openings and an accompanying case. The "sleeve" measures approximately 12 inches in diameter and 3 feet in length. Up to three animal cages (11 " x 7 " x 5") can fit in the sleeve. To move animals from an isolator to a germfree shipper takes careful maneuvering. One end of the shipper sleeve is connected to the isolator and empty animal cages (pre-positioned in the shipper) are carefully moved from the shipper to the isolator.One end of the shipper sleeve is connected to the isolator and empty animal cages (pre-positioned in the shipper) are carefully moved from the shipper to the isolator. The mice located in the isolator are placed into the cages in accordance with prescribed densities. The cages are then moved back into the germfree shipper, which is then disconnected from the isolator and sealed. The animals are never exposed to outside air; only filtered isolator air and then air from the filtered opening of the germfree shipper. Once animal cages have been placed in the germfree shipper, the shipper is placed in a cardboard box (or a clear plastic box for foreign shipments) for transport to its destination. The Taconic germ free shipper density is mentioned below: 24 at 3-4 weeks (8 per cage) 21 at 5 weeks (7 per cage) 21 at 6 weeks (7 per cage) 18 at 7 weeks (6 per cage) 15 at 8 weeks and older (5 per cage) Removal of Mice From the Germ Free Shipper: 1. Withdraw vinyl shipper from the cardboard box (use care to keep cages and oval filter upright). 2. Open end of connecting sleeve: a. Unfold sleeve and remove yellow vinyl tape that was folded and sealed prior to shipment. b. When open the sleeve is compatible with a standard 12" diameter Isolator port.

3. Swab inside of sleeve and the face of the Mylar diaphragm with a sterilant (using caution not to apply too much pressure to Mylar diaphragm). 4. Pull sleeve partway onto the swabbed port of the destination Isolator, spray sleeve portion of shipper with sterilant by puckering the sleeve around the nozzle of the atomizer and spraying until the sleeve is inflated. 5. Secure with tape, to make a complete tape seal of the shipper to the destination Isolator port. 6. When ready remove the yellow tape from the circumference of the Mylar diaphragm. 7. Carefully, work the Mylar diaphragm to a horizontal position (using external manipulation). 8. Pull the diaphragm through the port and into the Isolator. 9. Rotate the cages and slide toward the Isolator port (using external pressure). 10. Pull in cages and remove the bands that are securing the cages lids and remove animals. Avoiding Contamination of Animal Facility Via Shipping Boxes: For this purpose we should carry out the following y y y y Inspection of boxes on arrival at the animal facility. Decontamination of the boxes exterior. Unpacking boxes in animal Rooms. Preparing used boxes for recycling.

Decontamination of Boxes: In order to decontaminate the boxes the following sprayers are used: Hand Sprayer: It is used for spraying 1-6 boxes. Pump Sprayer : It is the sprayer with adjustable nozzle for spraying more than ~ 6 boxes. Proper method of spraying is critical and no area of the exterior of the box should be missed.

Quality Control of SCID mice:

The SCID mice are maintained as a true congenic strain. Each production isolator has a foundation colony from which animals are selected for breeding. These breeders are used to produce offspring for sale. A primary foundation colony is also maintained in a separate isolator and is used to restock the satellite foundation colonies within individual production isolators. All foundation colonies are pedigreed. The Prkdc scid mutation is recessive and all of the production health monitoring program ensures the animals health status. Immunocompetent female sentinel mice(pigmented strain to avoid unnoticed accidental interbreeding) are removed from each isolator and bled for serologic screening for adventitious viruses and mycoplasma. SCID mice are removed from each isolator and undergo a complete health monitoring exclusive of serology. This includes gross pathology, histopathology as required parasitology and bacteriology.At each complete health monitoring animals are assessed for the presence of Pneumocystis.murina . In addition to test for common human borne bacteria such as Staphylococcus.aureus , microbiologic culture of each individual isolator is conducted monthly. On a regular basis animals are removed from each production isolator and tested for IgG and IgM levels for any incidence of leakiness.