Mice.

Laboratory Mouse Mus musculus Outbred stocks and inbred strains
Order Rodentia, family Muridae Outbred stocks and inbred strains Adults weigh between g on average Variety of colors Black strains have black eyes and coat Albino strains have white coats and pink eyes

Genetic Categories Outbred stocks Inbred strains F1 hybrids Trangenics

Genetic Categories Outbred stocks Maintained in large populations
Genetic composition of population remains stable Unique, heterogeneous, Matings planned to minimize genetic changes Examples Swiss Webster, CD-1, ICR

Genetic Categories Inbred strains Nearly genetically identical
20 generations of planned brother x sister or parent x offspring matings 99% or greater homozygosity Produced to select for specific trait Common strains C57BL BALB/c C3H

Genetic Categories Inbred strains Common mutant strains
Nude (nu/nu) Deficient in T-cell lymphocytes Nearly hairless Severe combined immunodeficient mice (scid/scid) Deficient in both T-cells and B-cells Nudes and SCIDS are used in oncology research Accept transplanted human and murine tumors

Genetic Categories F1 Hybrids
First progeny of mating between two different inbred strains Parental sex from each strain must be consistent Genetically identical at all loci Hybrid vigor Common hybrids B6D2F1 CD2F1

Genetic Categories Transgenic technology
founded on ability to alter genetic makeup of an organism DNA orchestrates the function of an organism Nucleotides→ DNA→ Cell→ Organ→ Organ System→ Organism

Transgenic Mice Foreign piece of DNA (transgene) integrated into their genome DNA can originate from mouse or other organism DNA sequence can remain same or can be modified by researcher

Microbiologic Classifications
Germ-free (axenic) Free of all detectable microflora Gnotobiotic Known associated microflora Specific pathogen free Free of a defined list of pathogens Conventional Undefined microflora

Eliminating Unwanted Research Variables
Institutional operational procedures Examine health status of animals received from other institutions or commercial vendors Mice that are infected with a known pathogenic agent can undergo cesarean rederivation to obtain pathogen-free pups

Uses #1 mammal used in research Valuable attributes
Reproduction, teratogenicity, genetics Toxicity and carcinogenicity studies Valuable attributes Short life span Short gestation Large litter size Genetic diversity Anatomy and physiology well characterized

Behavior Easy to handle Curious and social Build nests
Tend to be territorial Males frequently fight Certain strains e.g., SJL, are prone to aggression Bite wounds May be fatal

Behavior Barbering Abnormal behavior
Removal of hair and whiskers from faces, heads, and bodies of other mice Distinct line of demarcation between the bald and haired areas Skin has no wounds Both dominant and subordinate mice barber More common in females

Anatomic and Physiologic Features
Small bodies covered in soft, dense fur Short legs Long, thin, hairless tails Dental formula 2 (I 1/1, C 0/0, P 0/0, M 3/3) Incisors Grow continuously throughout life Worn down by abrasion of occlusal surface

Stomach Divided into nonglandular forestomach and glandular stomach Brown fat Important in nonshivering thermogenesis Mammary glands 5 pairs Widely distributed extending onto sides and back

Open inguinal canals Males have os penis Sexing Genital papilla more prominent in male Distance between anus and genital papilla is 1 ½-2 times greater in the male

Hematology Lymphocytes are predominant circulating leukocyte Basophils rarely found Site of collection, time of day can influence WBC counts of limited diagnostic value Urine Excreted one drop at a time Highly concentrated Large amounts of protein pH

Breeding and Reproduction
Continuously polyestrous Estrous cycle is 4-5 days Can become reproductively active shortly after weaning Sexes should be housed separately to avoid unwanted pregnancies Sexual maturity 6-8 weeks of age 20-30 g

For maximum production, breeding should begin shortly after sexual maturity be continued throughout breeding life of female Systems Monogamous One male, one female Defined mating pairs must be maintained Polygamous One male, multiple females “trio” (one male, two females) commonly used Maximizes production and space utilization

Pheromones Chemical substances secreted from body that elicit a behavioral reaction in recipient Important in mice Whitten effect Synchronization of estrus Large groups of females housed together, then exposed to male odor Bruce effect Aborting pregnancy Recently bred female mouse exposed to strange male Lee-Boot effect Female mice housed in group without male exposure become synchronized in estrus With absence of male pheromones, females have suppressed estrus cycle

Adults and offspring may be co-housed until weaning Reproductive performance decreases with age Breeders replaced at 8-10 months of age Mating is confirmed by presence of Sperm in female Vaginal plug Gestation days

Litter size Strain and age dependent Between 6-12 pups Pups Altricial Fur covering and ears open by day 10 Eyes open by day 12 Begin eating solid food at 2 weeks of age Weaned at 21 days Co-house with other pups or provide nesting material to support body temperature

Husbandry Housing and Environment Shoebox-style cages
constructed of durable plastic (polycarbonate, polypropylene, polysulfone) Fitted metal lid consisting of parallel bars Depression in lid accommodates food pellets Divider in lid separates feed from the designated water bottle area

Husbandry Housing and Environment
Solid bottom caging with bedding recommended Wire bottom cages should be strongly avoided Increased limb injuries Less comfortable to animal than solid bottom Hinders temperature self-regulation

Housing and Environment
Microisolation cages (MI) Commonly used to house laboratory mice, especially immunosuppressed animals Shoebox-style cages made of durable plastic; have plastic lids with filters Airflow into and out of cages occurs through passive diffusion Results in higher humidity and build up of ammonia

Microisolation cages (MI) Ventilated caging systems Cages attached to rack via a ventilation port Allows for individual cage ventilation and protection from airborne contaminants Decreased frequency of cage changing Helps to contain allergens Single use disposable caging Beneficial in studies when animals are administered a substance hazardous to personnel Used to transport animals short distances

Space requirements found in the Guide Depends on number of mice housed in cage Additional floor space needed for Females with litters Larger mice 5 inches minimum cage height Cage must have a secure cover Mice that escape do not return to the cage Contact bedding material placed at bottom of cage Hardwood chips, composite recycled paper pellets, corncob particles

Rigid control of room temperature, humidity, ventilation, and lighting is essential 680 to 790F 30% to70% humidity 10-15 fresh air changes per hour in animal room 12-14 hours of light per day, controlled through use of automatic timers

Environmental Enrichment and Social Housing
Guide places increased emphasis on environmental enrichment and social housing Mice are social animals Do best when housed in small, compatible groups Enrichment items may be of benefit Nesting materials Items that animal can manipulate or gnaw Increased cage complexity Huts Running wheels Enrichment items can produce unintended effects Aggression Traumatize eyes and feet of nude mice

Feeding and Watering Mice consume 4-5 g of solid food per day
Complete commercial rodent diet best Pelleted form fed ad libitum in elevated hoppers 16% protein and 4-5% fat Mice are coprophagic Ill mice can benefit from a container of moistened or gelatinous chow placed on cage floor Feeding powered or soft chow can lead to increased incidence of malocclusion

Feeding and Watering Feed should be
Fresh Stored in a cool, dry location (<700 F) Used before expiration date Open bags of food should be stored in a closed container

Feeding and Watering Adult mice drink 6-7 mL water per day
Water should be Fresh, potable Available ad libitum through water bottles with sipper tubes, sipper sacks or automatic watering systems Weanling and recently shipped mice should be closely observed for dehydration

Sanitation Frequency of cage cleaning depends on
Number and size of mice housed Amount of air movement in the room or cage One to two times weekly is usually adequate Ventilated cages can be changed less frequently

Sanitation Cages, water bottles, and feed hoppers should be disinfected with Chemicals Hot water (1430 – 1800 F) Combination of chemicals and hot water Detergents and chemical disinfectants enhance the effectiveness of hot water, but they must be thoroughly rinsed from caging surfaces Residual water should be removed from rack manifolds before placement into the rack washer

Techniques Handling and Restraint Exam gloves should be worn
To transfer mouse from cage to cage Pick up by base of tail with fingers or rubber-tipped forceps To restrain Grasp base of tail with thumb and forefinger of dominant hand Place mouse on surface to which it will cling Scruff loose skin at back of neck between thumb and forefinger of non-dominant hand Lift mouse and secure tail between the pinky finger and palm of same hand Mechanical restrainers are commercially available Allow for access to blood collection and injection sites

Techniques Identification
Cage cards used as general means of identifying caged mice Individual mice Ear punch Ear tag Ear or tail tattoo Subcutaneous microchip Fur dye (temporary)

Techniques Blood collection Sites
Up to 10% of circulating blood volume (1% of body weight) can be withdrawn every 2-4 weeks Larger volume or more frequent collection is possible if fluid replacement is provided Sites Retroorbital sinus (requires anesthesia) Lateral saphenous vein Facial vein Cardiac puncture (requires anesthesia, normally terminal procedure)

Techniques Genotyping
Used to characterize the genetic makeup of breeding or genetically modified mice Sample of DNA Taken from distal tip of tail Pups 21 days of age or younger, no anesthesia required Mice greater than 21 days of age, anesthesia required Ear punch tissue can also be used Toe amputation is strongly discouraged

Techniques Urine Collection
Mice can be stimulated to urinate upon handling or placement on cold surface Use of metabolic cage Daily volume production of adult is mL

Techniques Drug administration Obtain accurate body weight
To increase dosing accuracy Dilute drugs Use syringes designed for small volume administration Calculate dosing volumes on a mL/kg basis

Techniques Oral administration Injectable Drug mixed in feed or water
Specialized gavage needles Use appropriate volume when giving by syringe or gavage Injectable Smaller needles (25g) should be used Subcutaneous under loose skin over shoulders

Techniques Injectable Intramuscular injections not preferred route
Intraperitoneal Administer in lower abdominal quadrant just off midline Best route for ketamine and other irritating drugs Intravenous Use of tail vein

Anesthesia, Surgery, and Postoperative Care
Dilution of all injectable agents is recommended Age, sex, strain, health, and body condition influences the dose needed Ketamine and xylazine combination is a good injectable regimen for surgical procedures Inhalation anesthetic agents can be used with precision vaporizer Isoflurane Sevoflurane

Rodents are no more resistant to infection than other mammals Aseptic technique should be used Multiple rodent surgeries Autoclaving several small packs Use of hot bead sterilizer Use of one pack for small number of animals (up to 5)

Intra and Postoperative Care Prevention of hypothermia is key Circulating water blanket Medical grade heat pack Use sterile ophthalmic ointment if animal’s blink reflex is lost Provide subcutaneous fluids to animals who have undergone prolonged surgeries to prevent dehydration

Pain control Analgesics must be administered to control surgical pain Any procedure that is known to cause pain to humans must be assumed to cause pain in animals Signs of pain Difficult to assess Decreased food and water intake Decrease in activity Piloerection Analgesics Nonsteroidal anti-inflammatory drugs (meloxicam, ketoprofen, carprofen) Opioids (butorphanol, buprenorphine)

Anesthetized mice Should be placed on a dry paper towel (not small particle bedding) in a safe area for recovery Should not be placed with animals that are not anesthetized until they are fully awake Circulating water blanket placed under half the cage or heat lamp directed at a corner of cage can prevent hypothermia Provision of moistened chow or a gelatinous water or nutrition source can assist in recovery

Imaging Techniques Many modalities have been adapted to use in small rodents Digital x-ray MRI CT PET DEXA Faxitron

Radiography X-ray machine capabilities Restraint
High milliamperage ( mA) Low kVp settings Short exposure times (1/40 or faster) Restraint Sedate patient Use of tape, radiolucent tube, stockinette for positioning

Euthanasia Provide rapid death with little to no pain or distress
Personnel must be properly trained Methods must be consistent with the AVMA Guidelines on Euthanasia CO2 commonly used Gas tank with flow gauge Mice are exposed to controlled displacement of air

Euthanasia Fetal mice Neonates
Under 15 days of gestation may be euthanized by allowing them to remain in utero following euthanasia of dam Removed from the uterus and allowed to breath air, must be promptly euthanized by a physical method Neonates Under 10 days of age show resistance to CO2 Need prolonged exposure to CO2

Euthanasia Inhalant anesthetics Physical methods
Performed under anesthesia Cervical dislocation Decapitation Methods to ensure death Bilateral pneumothorax Removal of a vital organ Exsanguination by transection of a major vessel

Humane Endpoints Animal may require premature removal from a study due to impaired physical condition or imminent death Discrete criteria used to determine when euthanasia should occur Loss of portion of body weight Inability to eat, drink, or ambulate Loss of consciousness Large tumor mass

Transgenic Mice Production
Two methods used to create Pronuclear injection Injection of DNA into fertilized eggs Gene targeting Injection of genetically modified embryonic stem cells into preimplantation embryos

Pronuclear injection Foreign DNA containing gene of interest is injected into the pronuclei of a fertilized egg FVB strain used as pronuclei are large, produce large number of embyros, and are good mothers Multiple fertilized eggs, each injected with the foreign DNA, are surgically implanted into a recipient female Following birth, tissue sample tested from each pup to identify animal with foreign DNA (founder animal) Each founder animal is backcrossed to an inbred strain to produce unique mouse strain

Gene targeting 129 and C57BL/6 strains commonly used Preimplantation embryo is harvested from a donor female Embryonic stem cells are mixed with DNA segments containing the transgene and the culture environment is manipulated to allow DNA insertion Genetically modified ES cells are screened to select those that have DNA inserted ES cells with genetic mutation are injected into a host preimplantation embryo and mix Embryo is surgically implanted in a surrogate, pseudopregnant dam Chimeras (possess tissues derived from genetically modified ES cells) are identified and bred, selecting for the desired mutation

Gene Targeting Knockout (KO) mice can be engineered Knock-in (KI) mice
Specific gene is deleted or disrupted Gene function can be determined by examining the abnormalities produced Knock-in (KI) mice Transgene is inserted in a specific location without deletion or inactivation of the normal genetic sequence

Transgenic Mice Health complications can arise
Compromised immune function Decreased reproductive ability Altered growth patterns Painful musculoskeletal conditions Original mouse strains utilized to create a transgenic animal can influence phenotypic expression and disease conditions that develop

Introduction to Disease
Improved environmental controls and husbandry have led to identification and eradication of many infectious agents Most rodent colonies are relatively free of viruses, bacteria, parasites, and fungi that cause clinical disease Sporadic outbreaks do occur Evaluation of clinical signs in animal is difficult Serology, histopathology, and microbiology often needed for definitive diagnosis

Introduction to Disease
Introduction of pathogens into facility Personnel Equipment Infected animals Biologic materials Increased risk through sharing of rodents and tissues nationally and internationally Use of immunocompromised and genetically altered strains of mice is on the rise Population is more susceptible to disease

Bacterial Diseases Pneumonia and Respiratory
Uncommon unless animals are immunologically deficient or stressed Mycoplasma pulmonis, cilia-associated respiratory bacillus Clinical signs Teeth chattering Labored respirations Weight loss Conjunctivitis

Bacterial Diseases Helicobacter Infection Numerous strains
Inhabit GI tract from stomach to colon in variety of species Transmission occurs by fecal-oral route Infections are persistent Mice vary greatly in their susceptibility to infection Helicobacter species vary in pathogenicity

Bacterial Diseases H. bilis and H. hepaticus documented pathogens
Associated with disease syndromes Chronic active hepatitis Hepatic neoplasia Inflammatory bowel disease Proliferative colitis Rectal prolapse Infected mice should not be used in long term studies or housed near immunodeficient animals Immunodeficient and aged animals have higher incidence of hepatic and GI lesions and tumors

Bacterial Diseases Helicobacter
Clinical signs normally not seen until liver disease is end stage Sentinel mice exposed to dirty bedding can be used to screen for presence Polymerase chain reaction (PCR) testing of fecal pellets or intestinal tissue samples most accurate and sensitive tool available for diagnosis Multi-antibiotic regimen can be used to treat Eradication achievable by transferring neonates to clean foster mother and rederivation

Bacterial Disease Tyzzer’s Disease Caused by Clostridium piliforme
Spores can remain stable for up to 1 year Spores are likely means of transmission through contaminated food and bedding Occurs more frequently and is usually more severe in recently weaned animals, immunosuppressed animals Clinical signs include diarrhea, dehydration, anorexia Gross lesions seen at necropsy include miliary, pale foci throughout the liver Tetracycline used to treat

Bacterial Diseases Transmissible murine colonic hyperplasia
Caused by Citrobacter rodentium Clinical signs include diarrhea, retarded growth, ruffled fur, soft feces Mortality rate is variable Characteristic gross finding at necropsy is thickening of distal half of colon PCR suggested for diagnosis

Bacterial Diseases Hyperkeratosis-Associated Coryneform (HAC)
Caused by Corynebacterium bovis Infection is referred to as scaly skin disease Occurs in nude mice of all ages and neonatal SCID mice High mortality in suckling mice Clinical signs include dry, white flaky skin, dehydration, and pruritis Transmission is by direct contact People can serve as source of infection for mice No known treatment, affected animals should be euthanized to prevent spread

Bacterial Disease Miscellaneous Staphylococcus aureus and S. xylosus
Commonly affects skin Associated with abscesses Streptococcus spp. Associated with dermatitis, pharyngitis, cervical lymphadenitis, bacteremia Pseudomonas aeruginosa Contaminated, untreated drinking water is the most common source Opportunistic organism Frequently isolated from preputial gland abscesses Mice that are irradiated or immunosuppressed are susceptible

Bacterial Disease Miscellaneous Corynebacterium kutscheri
Produces a disease in stressed or immunosuppressed mice called pseudotuberculosis Characterized by septicemia with high mortality Produces disseminated abscesses Treatment is usually not successful Salmonella spp. Rare problem Infection is significant due to its ability to infect and cause disease in humans Streptobacillus moniliformis Not common One of the causes of the zoonotic disease rat-bite fever

Viral Diseases Parvoviruses Small non-enveloped DNA viruses
Can persist in the environment for months to years Two parvoviruses known to naturally infect laboratory mice and are among most prevalent infectious agents found in colonies Mouse parvovirus (MPV) Mouse minute virus (MMV) Neither typically causes clinical disease

Viral Diseases Parvoviruses Target intestinal and lymphoid tissue
Differences in strain sensitivities to infection MPV is of greater concern Causes immune dysfunction Can infect and persist in lymphoid tissue Transmission is slow and thought to be fecal-oral Adult mice and neonates are susceptible to infection Diagnosis is by serological screening of sentinel mice or by use of PCR technology Depopulation of infected mice and/or cesarean rederivation is used to control

Viral Diseases Sendai Virus Highly contagious Parainfluenza 1 virus
Rarely seen Can cause clinical respiratory disease Distinct strain variation in susceptibility to infection Immunosuppressed mice are highly susceptible and develop a wasting disease Aerosol transmission Recent outbreaks are associated with contaminated biologic materials Self-limiting infections in immunocompetent animals

Viral Diseases Mouse Hepatitis Virus (MHV)
Highly contagious coronavirus One of the common viral contaminants Virus shed in feces and respiratory secretions Transmission occurs thorough direct contact, fomites, and airborne particles Many related strains that fall into two disease patterns Enteric (most common) Respiratory

Viral Diseases MHV Infections are usually enzootic and subclinical
Epizootics occur in naïve suckling mice Clinical signs Severe diarrhea Runting Empty stomach Encephalitis with tremors High mortality Infection in nude mice is associated with a severe wasting disease

Viral Diseases MHV Diagnosis through ELISA or PCR testing
Distinctive histologic finding Syncytial cells in the small intestinal mucosa and liver parenchyma Virus does not persist in immunocompetent mice Can be eliminated from a colony by cessation of breeding and no introduction of susceptible mice for at least 4 weeks

Viral Diseases Epizootic Diarrhea of Infant Mice (EDIM)
Rotavirus that causes disease in suckling mice less than 2 weeks of age Clinical signs Soft, yellow feces or accumulation of dried feces around the anus Affected mice continue to nurse Diagnosis is usually by ELISA serology Use of microisolation cages can prevent spread No treatment other than supportive care

Viral Diseases Murine Retroviral Infection
All mice harbor endogenous proviruses in their genome Murine leukemia virus (MuLV) Murine mammary tumor virus (MuMTV) Genetic transmission Expression of disease is strain-, age-, and cell-type-specific

Viral Diseases Ectromelia Poxvirus Uncommon in the US
Disease is introduced through imported mice, tumor transplants, or other biologic materials High morbidity and mortality Clinical signs include conjunctivitis and pox-like rash Severe infection of feet and tail can led to amputation (ectro = loss; melia = limbs)

Viral Diseases Pneumonia Virus of Mice (PVM)
Pneumovirus that replicates in the respiratory tract Asymptomatic infections May cause wasting disease in immunodeficient mice Diagnosis is by ELISA screening of sentinel mice Self-limiting infections

Viral Diseases Murine Norovirus (MNV)
Noroviruses are important human pathogens, causing more than 90% of nonbacterial epidemic gastroenteritis Many strains detected Common viral infection in laboratory mice Affects the immune system Immunodeficient mouse strains have high mortality with encephalitis, pneumonia, and hepatitis

Viral Diseases Miscellaneous Mice are susceptible to many viral agents
K virus Mouse polyoma virus Reovirus type 3 Theiler’s encephalomyelitis virus Mouse adenovirus Lactic dehydrogenase elevating virus Rarely cause disease in immunocompetent mice

Parasitic Diseases Mites Sporadically detected
Myobia musculi, Radfordia affinis, and Myocoptes musculinus most common Degree of pathogenicity is highly variable Some transgenic strains are especially sensitive Infestation can induce immunologic changes

Parasitic Diseases Mites
Diagnosis can be accomplished by skin scrapings, fur plucks, applying cellophane tape to fur PCR assays have been developed Eradication Topical ivermectin therapy Surgical rederivation most reliable

Parasitic Diseases Pinworms
Syphacia obvelata and Aspicularis tetraptera inhabit the cecum and colon High portion of conventional colonies affected Clinical signs are usually absent Can alter the humoral immune response Strains differ in their susceptibility to infection Not transmissible to humans

Parasitic Diseases Pinworms
Premortem diagnosis can be challenging as eggs are intermittently shed Diagnosis is by fecal exam or cellophane tape to perianal area (Syphacia) Examining cecal contents postmortem for adult worms is most definitive Treatment includes ivermectin, fenbendazole, piperazine

Parasitic Diseases Lice Tapeworms Highly uncommon in laboratory mice
Rodententolepis nana and Hymenolepis diminuta Most cases asymptomatic R. nana presents a public health concern as an intermediate host is not needed for human infection

Parasitic Diseases Flagellates
Spironucleus muris and Giardia muris occur in small intestine and cecum Clinical signs Young animals - diarrhea Older animals - asymptomatic Metronidazole used to treat giardiasis

Neoplasia Incidence and types of tumors are highly strain- and age-related Retroviral infections play key role in development of lymphoreticular, hematopoietic, and mammary gland neoplasia

Neoplasia Mammary tumors Lymphoma Most are adenocarcinomas
Can produce large, firm masses on abdomen, sides or back High incidence in C3H strain Lymphoma High incidence in AKR strain

Bite Wounds Some strains are prone to aggression
BALB/c, SCID, SJL, and FVB Aggression most often seen in males Housing males together prior to weaning can decrease aggressive behavior Common wound locations Tail and perineum Back, shoulders, and head

Bite Wounds Wounds frequently become secondarily infected
Stress from fighting impacts the physiology of mice Treatments are usually ineffective Euthanasia is indicated in severe cases Addition of enrichment may be helpful in reducing aggressive behavior Enrichment items should be carefully evaluated as they can increase the aggression behavior Aggressive mice should be identified and removed from cages

Ulcerative Dermatitis
Genetically linked skin syndrome Frequently found in C57BL/6 mice or strain on C57BL/6 background Lesions most common over dorsal cervical and scapular regions Treatments have limited therapeutic efficacy Some success has been reported with trimming affected animal’s nails early in the disease process

Dehydration Cause of acute death Water may be unavailable
Air lock in sipper tube of full water bottle Malfunction of automatic drinking valves Mice may be slow in learning how to use a novel watering system Treatment involves SC or IP fluid therapy

Hair Loss Bilaterally symmetrical areas of alopecia on muzzle caused by friction from bars on the feeder Barbering behavior

Malocclusion Overgrowth of incisors can lead to emaciation and death
Excessive drooling of saliva with matting of fur around the chin and neck is a common clinical sign Incisors can be trimmed with Suture removal scissors Dental bur Some malocclusion has genetic basis Animals should be culled from breeding population

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