Back of Blue Book Flashcards
Approach for retro-orbital bleed in mouse/hamster/gerbil vs rat
Lateral approach (orbital sinus)
Direct the tube in a caudal
and medial direction through the dorsal conjunctiva (orbital plexus)
Topical vasodilators
EMLA
Limonene
Oil of wintergreen
How retro-orbital sinus bleed differs in mouse vs hamster
Both lateral approach, but hamsters orthogonally medial vs mice rostromedial
Chinchilla blood collection site under anesthesia
Transverse sinus medial to auditory bulla
Which mammals can blood be repeatedly drawn from heart?
Gray, short-tailed opossum (Monodelphis domestica)
Common opossum (Dilphis marsupialis viginiana)
Which side should vena cava be bled in birds?
Right vena cava usually larger; collection site of choice for Japanese quail (Coturnix japonica) - hematomas less common than with brachial vein of wing
Also right side in swine because of recurrent laryngeal nerve
Vascular Access Ports - type of needle to use, where to place, what locking solution
Huber needle (non-coring)
Tunnel to neck to reduce likelihood of animal traumatizing it
Anticoagulant
Most common site IO
medial tibia
System to collect bile in a dog vs larger species
T-tube biliary catheter system
Larger species: endoscopic retrograde cholangiopancreatography
2 types of intestinal loop isolation
Thiry fistula – one end exteriorized
Thiry-Vella loop fistula – both ends exteriorized
Preferred technique for anastomosis of intestine to intestinal wall
simple, interrupted, approximating suture pattern that limits intestinal tissue trauma and minimizes luminal narrowing
Name of technique to temporarily occlude a section of bowel for bacterial colonization studies in rabbit
RITARD (reversible intestinal tie-adult rabbit diarrhea)
Urinary catheter in male of this species becomes occluded due to ejaculation when it is passed
Guinea pig
Which species has highly friable ureters and get intraluminal bleeding with catheterization? How to mitigate?
Rabbit
Perfuse heparinized saline to flush out the blood
In which species can the proximal urinary tract be readily accessed by retroperitoneal approach?
Swine; used to model conditions affecting ureter and urinary bladder
Can transplant ureters into intestine or Heidenhain pouch (denervated pouch generated from stomach)
What technique is used to model renal fibrosis in mice?
Ureteral obstruction –> ipsilateral renal fibrosis
What is tracheal pouch formation used for?
To study tracheal secretions e.g. NANCI in guinea pig (nonadrenergic non-cholinergic inhibitory neurotransmission)
What volume of saline to use for BAL of dogs/swine, children, and human adults? How to do it and how many wash sequences to do?
Dogs and swine: 50 to 300 ml
Children: 3 ml/kg
Adult human: 10–300 ml
Hyperventilate –> instill warmed saline –> immediately withdraw until feel slight resistance
3-4 wash sequences = adequate sampling
How to create pneumoperitoneum for laparoscopy?
Veress needle or cannula attached to an insufflator, abdomen inflated w/ 5% CO2 or 5% nitrogen
Name of positions for laparoscopic procedures of 1) lower abdomen/pelvic and 2) upper GI
1) Trendelenburg (head down)
2) Reverse Trendelenburg
Which castration technique to use to study reproductive physiology?
McGlinn’s technique - remove seminiferous tubules and Leydig cells, leaving epididymis and nerve/vascular supply intact
For species with open inguinal canal, also suture tunica albuginea and scrotal skin
How to determine female receptive to artificial insemination? How many viable sperm are needed?
Presence of cornified cells on vaginal smear (NHPs and many other spp)
At least 1 million (10^6) viable sperm
What is first palpable sign of pregnancy for NHP?
dorsoventral rounding of anterior aspect of the body of the uterus
Careful – abortion can occur if palpate before implantation; palpate at 30 days since onset of last cycle or use ultrasound which is gold standard to stage
Semen extenders / diluent extenders / cryoprotectants
dimethyl sulfoxide, ethylene glycol, glycerol, erythritol
What is the purpose of a polytetrafluorethylene (PTFE) graft?
Technique in larger species to anastomose the jugular vein and carotid artery to 1) generate a shunt for repeated blood collection and 2) increased afterload –> left ventricular hypertrophy
Can regular iodinated contrast agents be used in rodents?
No; they clear too quickly
Use special ones that bind to plasma proteins, although these have less contrast
Which species possess an almost flat sella turcica that positions hypophysis (pituitary gland) for rapid and easy removal? What approaches are available
Rodents
Hypophysectomy (removal of pituitary gland) approaches in rodents, dog, rabbit, calves
Rodents: Parapharyngeal approach has supplanted the transauricular technique
Dogs: extracranial route through oral cavity (transsphenoidal), intracranial route through anterior or middle cranial fossa and by the pharynrecess (=pharyngeal recess?)
Rabbit: paraocular
Calves: combination of transtemporal and parapharyngeal
What type of water source should be supplemented after 1) hypophysectomy 2) adrenalectomy 3) thyroidectomy/parathyroidectomy?
1) Glucose
2) Saline
3) 2% calcium lactate
In which species are parathyroids not always with the thyroid and care is needed to identify both for removal?
Guinea pig
In what disease is blood supply to the femoral head disrupted leading to avascular necrosis?
Legg-Calvé-Perthes Disease
Catheter-based strategies that interrupt ___ have shown promise for resistant hypertension
Renal sympathetic nervous system
Rabbits have been used to study ___ a condition where skull bones fuse prematurely and cannot expand
Craniosynostosis –> optic nerve compression, blindness and mental retardation
What are the two commonly used zero points for skull coordinates?
intersection of coronal and sagittal skull sutures (bregma) or the middle of an interaural line (line drawn between external auditory meatuses)
Two instruments used to remove periosteum from skull?
Bone curette
Periosteal elevators
What is the name of the scale used to measure coordinates for stereotaxic surgery?
Vernier scale
Which species have a well-developed sagittal crest that prevents the application of some types of headplates without modification?
Rabbits
___ mL of CSF can be collected per ___ kg of body weight.
1 mL per 5 kg
Which spaces to perform lumbar CSF collection in NHPs?
L4-L5 or L5-L6
Which species require surgical approach in stereotax for CSF collection? What volume is collected?
Mice (rats can be surgical or non-surgical)
Collect 7-8 uL for mice, 50-150 uL for rats
What is the in vivo Hollow Fiber Assay used for?
Implant multiple tumor lines into nude mice
What is main benefit of using zebrafish tumor models? How to achieve engraftment?
Shorter time to engraft than mice, more high throughput
Irradiate, administer corticosteroids, or administer before 48 hours when adaptive immunity develops
Disadvantage of intravesicular approach for transplanting bladder tumors?
Can seed along urinary tract
And in rats can also get renal because of vesiculo-ureteral reflux
What is the definition of microbiome?
collective GENOME of a host’s microbiota
What is the definition of microbiota?
COMMUNITY of microorganisms harbored by a host
3 main types of materials for gnotobiotic housing
1) Flexible plastic film made of PVC
2) Semirigid made of polypropylene
3) Rigid made of stainless steel
***2 and 3 can be operated at negative pressure for biocontainment
2 types of air outlet filters for gnotobiotic
3 does not gradually break down with repeated autoclave
1) Spun fiberglass
2) Pleated cartage-type
3) Spun polyurethane
How many air flow changes per hour for gnotobiotics?
At least 30 (vs 10-15 conventional housing)
Two types of transfer ports for gnotobiotics
1) Circular
2) Rectangular (requires circular adapter)
What are the most vulnerable parts of the GF isolator and most likely to fail leading to contamination?
GLOVES
How to use peroxyacetic or peracetic acid for sterilization of isolators
Prepare immediately before use; 30 min contact time
Use respiratory protection
What is Spor-Klenz? How to use it
Cold steriliant; combo 0.08% peracetic acid w/ 1% H2O2
Sporicidal and inactivates Mycobacterium
1 hour contact time
What is the most common liquid form of sterilant used in gnotobiotics? Name a downside
Chlorine dioxide (Exspor, Clidox-S)
1075x more sporicidal in gaseous state than ethylene oxide (ETO)
Sodium chlorite base solution + acidic activator 🡪 combined and mixed with water immediately before application to form soln of chlorous acid and chlorine dioxide
30 min contact time
Con: can corrode stainless steel (use Grade 316 contains molybdenum which enhances corrosion resistance)
What is in Steriplex SD?
silver 0.015%, peracetic acid 0.15%, ethanol 10%
Inactivates C. difficile and Bacillus subtilis spores!
What is the chief method of sterilization
of supplies and other materials in most gnotobiotic
facilities?
Steam!
Water content for steam sterilization should be no more than ___%. Most common temp is ___ or ___C.
3%
121C or 132C
What is the term for the test that uses thermochromatic (temperature sensitive) paper to test the efficacy of steam autoclave?
Bowie-Dick test
What biological indicator is used for autoclaving QC?
10^6 Geobacillus stearothermophilus spores test and validation of sterilization procedures 🡪 culture @ least 48h
Include in every cyllinder!
___ kGy is the minimum reported for gamma irradiation for sterilization. ___ kGy is the maximum allowed in US.
30-50
but there is no standard!
2 options for watering for gnotobiotics
1) Hydro pacs
2) Sterilize w/ 1.2 µm, 0.2 µm and 0.1 µm filters and 5-10 ppm free chlorine in water
Transfer pouches through dunk tank
What materials are used for gloves in gnotobiotics
1) Chlorosulfonated polyethylene rubber (CSP, Hypalon)
2) Polyurethane
3) Layers of CSP and polyurethane
CHEAPER, less durable:
4) Neoprene
5) Nitrile rubber
Minimum thickness for gloves in gnotobiotics
15 mil (0.015 inches)
but 30 mil too thick
How often to replace gloves in gnotibiotics
Annually
What type of diet is better for autoclaving?
Extruded better than pelleted (less compact, steam can penetrate better)
What are the most heat-labile nutrients to make sure are maintained through autoclaving?
thiamine (B1), pantothenic acid (B5), pyridoxine (B6), cobalamin (B12), vitamin A and E, and vitamin K
(KABE)
What is put at the end of the cylinder for gnotobiotics?
the open end of
the cylinder is covered with polyethylene terephthalate
polyester plastic film (Mylar™ diaphragm)
What is likely cause for germ-free isolator contaminated by 1) spore-forming bacteria 2) non-spore forming, aerobic bac 3) Staph 4) fungi
1) poorly autoclaved food or bedding
2) gloves or inadequate contact of liquid sterilizer
3) glove damage
4) inadequately sterilized food or growth on filter material
What is fine, cotton-like growth in water bottles of gnotobiotics?
Water mold (Oomycetes)
Top ddx for germ-free mouse found dead
- Most likely cecal torsion
- Contamination
Necropsy all found dead animals
How to increase probability of success for rederivation of germ-free mice
progesterone is used to delay parturition in donor mice
e.g. Derivation is scheduled for morning of gestational d19.5 –>
Donors given 1 mg progesterone SC on d18.5
Which four pathogens can be transmitted transplacentally/vertically?
- LCMV
- LDV
- Pasteurella pneumotropica
- Mycoplasma spp.
What is success rate of embryos transferred into germ-free recipient?
50%
What is the suspected cause of poor reproduction in germ-free?
estrogen conjugated by liver for excretion –> Gut bacteria deconjugate allowing reabsorption –> LESS ESTROGEN, prolonged diestrus
What are the main bacteria in conventional mice?
Clostridium spp, Enterobacter spp, Lactobacillus spp (x2), Enterococcus spp, Bacteroides spp = “CELEB”
Purpose of Altered Schaedler’s Flora
8 bacteria to normalize cecal volume and restore reproductive performance of germ-free mice
T/F Gnotobiotic facilities routinely HEPA certify
air filters, in the manner of biological safety cabinets.
FALSE
What are the two most likely sources of contamination in germ-free isolators?
- Gloves
- Incompletely sterilized food
What bacterial shapes on Gram stain are indicative of contamination in gnotobiotic mice with ASF?
- Cocci
- Blunt-ended, spore-forming rods
ASF = G+ large tapered rods, G- slender spiral, G+ medium tapered rods
IL-10 deficient mice colonized with ___________ develop colitis.
Enterococcus faecalis
What is the only federal entity that advises on vaccination?
The Advisory Committee on Immunization Practices (ACIP)
Which requires animals being housed in CDC-registered space: animals exposed to select toxin in lab or select toxin brought into housing to expose animal
The second option - if select toxin administered in housing
What is considered critical PPE for working with sharps, infectious agents/toxins?
Gloves, respiratory protection
Definitions of cleaning and disinfection
Cleaning – removal of excessive amounts of gross contam caused by animal waste, dirt and debris
Disinfection – reduction or elim of unacceptable concentrations of microorganisms
What laws enabled DHHS and USDA to make list of select agents/toxins? What groups were created to implement?
DHHS: Antiterrorism and Effective Death Penalty Act of 1996 –> The Division of Select Agents and Toxins (DSAT) in the CDC
USDA: Public Health Security and Bioterrorism Preparedness and Response Act of 2002 –> Agriculture Select Agent Program (ASAP) under USDA’s APHIS
DSAT + ASAP = Federal Select Agent Program
What BSL level should be used for the Flaviviruses?
Yellow fever (Aedes): 3
Dengue (Aedes): 2
West Nile (Culex): 2
Marburg has 100% mortality in what four species?
African green monkeys, rhesus macaques, GP and hamsters
Which species is naturally infected with Hepatitis E and sheds large quantities of virus? Which group of humans are at high risk?
Hepeviridae
Swine
Pregnancy women - 25% case fatality
Noma (necrotizing stomatitis) and retroperitoneal fibromatosis are observed with which retrovirus?
SRV (Betaretrovirus)
Affects OWPs (NWPs resistant)
Which type of PTLV causes lymphoproliferative disease in OWPs?
PTLV-1
(Primate T Lymphotropic Virus, a Deltaretrovirus)
Murine typhus (Rickettsia typhi) is primarily a disease of ____ and is spread by the oriental rat flea ____, which also spreads ____.
Rats
Xenopsylla cheopis
Plague!
Humans with Rickettsial Pox (Rickettsia akari) present with ____
Eschar with a shallow ulcer covered by a brown scab
Individuals w/ ___ should not work with C. burnetti d/t prospect of chronic, relapsing infxn
valvular heart dz
___ should be considered as ddx for infected dog or cat bite in immunocompromised person with fever.
Capnocytophaga canimorsus
Cat bite fever (Bartonella henselae) causes ____ in immunocompromised individuals
unusual vascular growth on skin (bacillary epithelioid angiomatosis)
Zoonotic bacteria that causes high mortality in rainbow trout and tilapia (cichlids) on fish farms
Strep iniae
Zoonotic Strep
S. iniae (fish)
S. zooepidemicus (GPs)
S. canis
S. suis
S. moniliformis (rat bite fever)
What is the most common laboratory-associated infection worldwide? Is it reportable?
Brucella, esp B. canis
B. canis not reportable
B. melitensis, B. suis, B. abortus are
What causes Weil’s syndrome?
Lepto
The most severe form with hepatic/renal dysfunction, hemorrhage, circulatory collapse
To culture Lepto, need ____ in the media. What is another diagnostic that is most often used?
Long-chain fatty acids (LCFAs)
microscopic agglutination test used most often- >1:100 considered significant
What bacteria causes the autoimmune disease Guillain-Barre syndrome?
Campylobacter
___ are the source of Helicobacter ____ for humans (zoonotic!) esp immunocompromised ppl
Hamsters
cinaedi
Gastric helicobacter-like organisms (GHLOs) include which three organisms?
H. suis, H. felis, H. bizzozeronii
Due to Salmonella concern, turtles with a carapace (shell) length of less than ___ inches/ ___ cm and turtle eggs may not be imported for any commercial purpose.
4 inches (10.2 cm)
Only for educational/scientific purposes
How to kill amoebic cysts (Entamoeba histolytica)? How about Balantidium coli cysts?
10 ppm chlorine or heat to 50C
Chlorine does not work! Prevent fecal contamination of water.
How long does it take for Cryptosporidium oocysts to sporulate after excreted in feces?
Already infectious at time of excretion
3 transmission routes for Toxoplasma gondii
1) congenital (vertical)
2) ingestion of infectious tissues
3) ingestion of oocyts
Why does daily cleaning of litter pans prevent accumulation of infectious Toxoplasma oocysts?
Sporulate in 1-5 days
Ticks are rare in lab setting except for _____.
Rhipicephalus sanguineus (brown dog tick)
Which mite produces three bites on a person known as “breakfast, lunch, and dinner”?
Tropical rat mite (Ornithonyssus bacoti)
Mice infected with LCMV ____ get persistent infection while mice infected ____ get transient.
In utero
After birth
____ strain of Yaba-like disease virus causes benign cutaneous masses in the ___ and ____. In contrast Yaba Virus causes ___ masses.
Tanapox; epidermis and adnexa
Subcutaneous
Unlike other members
of the Bunyaviridae family, ___ are maintained
in vertebrate–vertebrate cycles without arthropod vectors.
hantaviruses
Old world Hantaviruses are responsible for ___, while New World Hantaviruses are responsible for ___. Ribavirin has been shown to be beneficial for ____, but not ____.
HFRS, HPS
HFRS, HPS
Hepatitis ___ is unique among the major hepatitis viruses in that animals are natural reservoirs not humans
E
Of the four genotypes of Hep E which two are human only and which two are zoonotic from animals?
four genotypes: 1&2 hu only, 3&4 zoonotic
Swine experimentally infected with Hep E can infect ___, and human Hep E can infect ____.
NHPs; swine
The typical incubation period for humans for LCMV is ___ to ___ weeks and for macacine herpesvirus 1 is ____ to ___ weeks.
LCMV: 1-3 weeks
Herp B: 2-5 weeks
PTLV-2 is only found in ___ (species) and PTLV-3 is only found in ___ (type of primate)
Bonobos (Pan paniscus)
African
_______ and ________ are the most common clinical signs of NWPs infected with measles virus.
Hemorrhagic gastroenteritis
immunosuppression
Which 4 retroviruses have documented NHP to human transmission?
SIV, SRV, STLV, SFV
What is the vector for Rickettsial pox (Rickettsia akari)?
Liponyssoides sanguineus
Which group writes the Occ Health guide?
NAS (National Academy of Sciences), which contains ILAR
Nested PCR specific for the ____ and then ____ can be used to detect Strep iniae in fish.
16S-23S ribosomal intergenic spacer and a chaperonin 60 (cpu 60) gene
What are the two causes of rat bite fever? Are they reportable?
Streptobacillus moniliformis or Spirillum minus
Not reportable
Control of human yellow fever is centered on ___ and ___.
eradication
of the Aedes aegypti mosquito
human vaccination
with the live-attenuated 17D vaccine strain
What four diseases in primate quarantine are reportable and in what timeframe?
Marburg, Ebola, Yellow Fever, monkeypox
Report within 24 hours
The most common cause of atypical mycobacteriosis in humans is ____.
M. marinum
Which antibiotic eliminates C. jejuni from the intestine of most infected patients within 72 hours?
Erythromycin
What Strep spp. has been recently transmitted from guinea pigs to their owners, resulting in clinical septicemia?
Guinea pig
Of the causative agents of rat bite fever, _____ cannot be cultured and must be grown in another animal and then viewed via _____, whereas ______ can be grown on 15% blood agar, but growth will be inhibited in the presence of ______.
Spirillum minus; dark-field microscopy
Strep moniliformis; sodium polyanethol sulfonate (SPS), commonly used to enhance bacterial growth on agar plate by inhibiting antimicrobial activity of blood (don’t use!)
Atypical Mycobacteria have optimum growth at ___ to __°C, so infection localizes to ___.
30–32C; skin
Xenotransplantation is now directly under the auspices of the ___, which
coordinate and participate with global groups such as the WHO
FDA Cell, Tissue and
Gene Therapies Advisory Committee (CTGTAC)
Which species has or have been ruled out as potential donors for human xenotransplant therapies by United States federal regulatory agencies due to concerns of xenozoonosis?
NHPs!
T/F OSHA has set permissible exposure limits for iso and sevoflurane
FALSE
What were the first inbred strains of mouse and rat? and who produced it?
DBA by CC Little in 1909 @ Bussey Institute at Harvard
PA rat by Helen Dean King @ Wistar Institute
6 methods to tell if mice are genetically identical (i.e. if contamination presentation).
1) Coat color can tell you if pigmented strain contaminating albino
2) Tail skin grafting - Needs to be same sex or female to male (male –> female issues with Y chromosome)
3) Mandibular measurements
4) Biochemical testing for isoenzyme forms (using blood, tissue, urine) - only need to test for 10
5) DNA! RFLP (Restricted fragment length polymorphism) by Southern Blot, PCR (Simple Sequence Length Polymorphism or SNPs)
6) WBC alloantibodies
Current genetic monitoring of mice detects ___.
SNPs
Jackson determined that 29 SNPs needed to differentiate 300 strains
When to use low vs high density SNP panels?
Low - general monitoring (i.e. for contamination)
High - genome scanning, gene and quantitative trait locus (QTL) identification
What does GQC stand for?
Genetic Quality Control
A strain is considered inbred after ___ generations of sibling matings, but residual heterozygosity is diminished after ___ generations.
20; 40
For isogenic strains (differ at single locus), backcross to inbred parental strain every ___ generations to maintain the background or refresh the colonies with ___.
10
Cryorecovered embryos
A strain is congenic after ___ generations of backcrossing. How to make this faster?
10
Marker (eg SNP)-assisted backcrossing
How to monitor transgenic alleles (i.e. result of microinjection and random DNA integration)?
Periodically monitor transgene copy number and expression
What should ideally be done for conditional alleles containing loxP sites that require cre-mediated recombination?
Subsequent breeding, assisted by a cre-specific PCR assay, to remove the cre
For F2 hybrids, should breeders or progeny be tested for GQC?
Progeny because they are not genetically identical
How are recombinant inbred strains created?
RI lines are developed by the mating of randomly
selected F2 hybrid progeny followed by sib mating x 20 generations
What is the collaborative cross? How does it differ from advanced intercross?
The Collaborative Cross is a panel of recombinant INBRED mouse lines developed from 8 genetically diverse founder strains (5 classical lab strains + 3 wild-derived strains).
Advanced intercross (AILs) are created by intercrossing offspring for many generations (beyond the F2 generation), usually starting from a small number of founder strains (often two, but sometimes more). NOT INBRED
How to monitor advanced intercross?
Not inbred - need periodic reconfirmation by high-density SNP analysis
To maintain outbred stocks, each year ___% of animals in the foundation colony are refreshed with animals from the outlying colonies. Periodically, __% of male breeders in outlying colonies are replaced by males from the foundation colony.
5%
25%
What two types of colonies are needed to maintain outbred?
1) Foundation colony
2) Outlying colonies
To maintain an outbred stock, The mating pairs at each generation are selected by use of a ___, coupled with ___.
random number program
the exclusion of any randomly generated sib matings
What year was the C57BL/6J mouse genome sequenced? What type of sequencing was used?
2002
Sanger
Sequencing just the exome (coding regions) is an example of ___ sequencing.
Selective
The process of establishing sibling breeder units at weaning and recording each mating by family lineage in colony management software/cage cards/charts is known as ___ colonies.
Pedigreed colonies
____ provide the breeders to expansion stocks that, in turn, send breeders to the distribution colonies.
foundation stocks (FS)
For genetic monitoring, ___-specific assays are better than generic assays.
Allele
Examples of high-density SNP panels for mice
- Mouse Universal Genotyping Array (MUGA)
- MegaMUGA
- Mouse Diversity Array
What database compiles a catalog of SNPs for mice?
Mouse Genome Informatics (MGI)
___ are often used as approximate controls for mutant stocks that are not fully congenic
F2 hybrids
After ENU (alkylating agent) mutagenesis in males, dominant mutations will show in generation ___ while recessive will show in generation ___.
G1
G3
(G = genetic modification, F = filial)
In mice ES cells can be aggregated with the ___which forms at ___ DPC (days post-conception) or injected into the ___ which forms at ___ DPC.
Morula 2.5
Blastocyst 3.5
Embryonic stem cells are derived from the ___ stage, where the cells are contained within a ____ surrounded by ____.
Blastocyst
Inner cell mass (totipotent cells)
Blastocoel
The mouse strain ___ is highly amenable to culturing ES cells, which is likely related to the tendency for them to get ___.
129
Teratomas
How many founder animals to generate following microinjection of nucleic acid to ensure a phenotype is not result of insertional mutagenesis?
several (≥3)
___% of Tg founders may be mosaic d/t integration of transgene concatamers at two-cell stage or later
10-30
Pros and cons of using lentiviruses (first was Moloney Leukemia Virus) for transgenesis?
Pros: 100% of animals are founders
Cons: Hard to control # of transgenes, biosafety concern (lentiviruses can infect human cells)
What is the term for this: Randomly inserted transgenes disrupt genes and carry selectable markers where only cells where the transgene integrates within an active gene express the marker, allowing identification of the disrupted locus.
Gene trapping
To select for ES cells where homologous recombination occurred rather than random integration, the payload DNA can contain a marker outside of the homology arms, usually the ___ which acts as a ___ selection marker.
Herpes Simplex Virus thymidine kinase gene
negative (bc expression results in cell death)
What are the two repair mechanisms for double-stranded breaks? Which one is usually a knockout and which is usually a knockin when used with CRISPR?
1) Non-homologous end joining (NHEJ) - no template and usually results in indels — KO
2) Homologous repair (HR) or homology-directed repair (HDR) - has template — KI
What is the endonuclease for zinc finger and TALENs? How many bp is recognized by each? Which one is context-dependent?
Fok1
Zinc finger: 3 bp sequence & context-dependent
TALEN: 1 bp
How are Fok1 (zinc finger, TALEN) and Cas9 (CRISPR) different?
Fok1 needs to be dimerized (two of them each binding different strand of DNA)
Cas9 as a single protein can cut both strands
Which mouse strain is commonly used for PNI due to large pronuclei?
FVB
___ inbred rat is homozygous at the agouti locus (A/A) and is the 1st rat from which pluripotent ES cells were isolated
DA (dark agouti)
Tg founder always ____ for transgene, whereas ES cells modified by homologous recombination are ___.
hemizygous
heterozygous
Inbred substrains formed when animals are separated after __ but before __ generations of inbreeding.
20
40
What do tm vs en mean in nomenclature?
“tm” = target mutation from ES cell homologous recombination
“en” =programmable endonucleases e.g. CRISPR
What are the three types of drug toxicity curves?
1) V or U shaped curve (minimal toxicity in TNZ)
2) Linear (increased toxicity with increased temp)
3) Skewed (increased toxicity with temp only above TNZ)
How does temp affect sex ratio in fish/turtles vs crocodiles?
Fish/turtles: Low temp (22 C) male high temp (31 C) female biased
Crocodile eggs are opposite (low temp= females, high temp=males)
Ringtail in rats when RH < ___% and temp < ____.
25%
Thermoneutral zone (TNZ)
NH3 and CO2 permissible exposure limits for 8h TWA (time-weighted average)
25 ppm
5000 ppm
Rodents, cats, dogs, NHPs can detect ultrasounds, which are > ___ kHz
20
Rodents have peak auditory sensitivities in range of ___ - ___ kHz,
while human ear most sensitive at ___ kHz.
30-60
2
What strains of mice and rats are susceptible to audiogenic seizures?
AKR, BALB/c, SJL, CBA, SM, RF, LP, 129, DBA/2
Sprague Dawley and Wistar – noise exposure as neonates
Also gerbils
Light contamination at night with as little as ___ lux can cause what by what mechanism?
0.2 lux
Increases growth of transplanted Morris hepatoma, NMU-induced mammary tumors, human xenografted MCF-7 human breast cancer cells
Inhibits melatonin secretion and its suppression of tumor linoleic acid uptake and metabolism to 13-hydroxyoctadecadienoic acid –> more uptake by tumor cells and conversion to 13-HODE –> increased epidermal growth factor receptor / mitogen-activated protein kinase (MAPK) –> CELL proliferation
What extrinsic factor alters cellular stress responses?
Radiation
Exposure to ___ (extrinsic factor) increases risks of ocular damage, immunosuppression, immune modulation and neoplasia.
UV-B
Electromagnetic fields also affect neoplasia development (e.g. housing near MRI)
What type of caging causes pododermatitis; also associated w/increased incidence/severity of urologic syndrome in AKR mice?
Wire floor
Which type of bedding contains isomeric mixture of linoleic acid derivatives (THF-diols) = endocrine disruptors?
Corncob
___ can have higher levels of endotoxin than __ and ___ bedding.
Paper
Corncob; hardwood chip
___ bedding can induce trauma that is exacerbated by use of running wheels.
Hardwood chip
Vibration is defined by ___ and ___.
Amplitude and frequency
The frequency that causes an object to vibrate more readily and often increases its amplitude of displacement is known as _____. How does anatomic location affect this?
Resonance frequency
Differs by anatomic location; in the rat, thorax (225-230 Hz) > head (75-80 Hz) > abdomen (27-29 Hz) all higher than in hu
Regarding continuous long-term exposure to low levels of vibration from IVCs, vibration was highest at ___ frequency where rodents more sensitive
lowest
In aquatics, ___ can affect embryos of both Xenopus and Danio causing heterotaxia (abnormal position of organs due to altered cytoskeletal dynamics and tight jxns in the early embryo), neural tube defects (Xenopus) and tail morphogenesis (Danio)
low-frequency vibrations
The main exposure to xenobiotics is through ___.
diet
Estrogenic compounds are mainly from ___ and ___ in the diet. What are the two main types of phytoestrogens?
Soybean meal, alfalfa
1) Isoflavonoids (e.g. genistein and daidzein), 2) coumestans
The main diet used in GLP/tox is ___, which minimizes exposure to xenobiotics.
Certified
High Vitamin __ is a teratogen in rabbits.
A
Steam autoclave can increase ___, a neurotoxin and carcinogen, in rodent diets by 14X.
Acrylamide
Gamma irradiation of cat diets can reduce Vitamin A and increase peroxides, leading to ___.
Leukoencephalomyelopathy
What happens when chlorine is used to disinfect water that has organic materials?
Reacts with bromide/iodide –> trihalomethanes (Carcinogen) e.g. chloroform
Which gas anesthetic is nephrotoxic and causes diabetes insipidus in F344 rats?
Methoxyflurane
Which anesthetics cause corneal calcification/keratoconjunctivitis sicca in rats?
Ketamine/xylazine
What is mechanism for hepatic toxicity of halogenated ether gas anesthetics? Which one is the worst?
Tissue acetylation –> neoantigens –> antibody-mediated immune response
Halothane
Compound A secondary to sevoflurane is highest with ___ flow rates, ___ sevo concentrations, and ___ absorbant.
Low flow rate
High sevo
Barium hydroxide (vs soda lime)
Neuroprotective anesthetics
dexmed, halothane, iso, ketamine, pentobarbital, propofol
↓ ___ and ↑ ___ due to xylazine
insulin
glucose
Effect of iso on WBC, neutrophil, and platelet counts?
DECREASE
Euthanasia by ___ is used to maintain integrity of enzymatically labile neurochemicals
Microwave
Procaine, used in penicillin formulations, can cause toxicity in __, __, and __.
Rabbits, guinea pigs, mice
Natural antimicrobial compounds include ___ in frog skin and ___ in bovine neutrophils.
Magainin
Indolicidin
What protein in vaginal secretions of hamsters triggers copulatory behavior in male hamsters?
Aphrodisin
In which behavior test can pheromones be retained causing problems?
Porsolt forced swim test – water may retain pheromones and affect rats subsequently tested
Increased colonic adenocarcinoma in mice infected with Citrobacter rodentium and administered ____.
1,2-dimethylhydrazine (DMH)
___-frequency sounds can alter several biologic parameters ex BP, water consumption, repro etc.
Low frequency sounds (≤ 20kHz)
Which anesthetic when given to pregnant mice, increased the incidence of microphthalmia ~8 fold
Buprenorphine
Which two anesthetics affect the circadian secretion of melatonin?
Ketamine, pentobarbital
Which xenobiotic can cause increases in thyroid-stimulating hormone and decreases in circulating T4
SMZ (sulfamethoxazole, commonly used to prevent Pneumocystis)
Which xenobiotic competes with GABA receptors and can interfere with studies involving the central nervous system?
Fluoroquinolones
What is a common viral and bacterial contaminant of cell lines?
MVM (parvo)
Mycoplasma
Mouse and rat spontaneous Hirschsprung models
Mouse: Piebald (Ednrb)
Rat: Spotting lethal
Which mice are a spontaneous model of autoimmune diseases?
NZW/NWB F1 hybrids
Athymic nude mice are similar to ___ syndrome in humans
DiGeorge
__ and __ have a mutation in the ___ and are a model of Chediak Higashi Syndrome
Mink, beige mice
Lyst gene
What animal is a model of esophageal achalasia (failure of LES to relax)?
Cats
What animal is a model of congenital ichthyosis leading to hyperkeratosis?
Cattle
Which animal is a model of Dubin-Johnson syndrome (hyperbilirubinemia due to excretion defect)?
Corriedale sheep
Chickens with ___ led to the cure for this in humans and animals and the discovery of vitamins
beriberi (thiamine deficiency)
Traditional rodent models
of toxicology studies include the ___ mouse and the ___ rat
B6C3F1 hybrid
Fisher F344
Which researcher used inbred mice to discover MHC as the cause of graft rejection in humans?
George Snell
__ are genes with a common ancestral gene that retain a similar function in different species, while ___ are genes related by duplication within the genome and often acquire new function.
Orthologs
paralogs
Gene duplicates that arose from whole-genome duplication known as teleost-specific duplication in fish are ___.
Ohnologues
Sets of SNPs residing on the same chromosome are inherited in blocks. The pattern of SNPs in a block is called a ____.
haplotype
Which coat color pattern in mice is controlled by DNA methylation?
Agouti
What is the term for conservation of the ORDER of genes between two species or chromosomes being compared?
Synteny
Tumors were reported in germ free mice exposed to bedding treated with which sterilization method?
Ethylene oxide
Pronuclear injection has low integration rates of about _____% for creating founder animals. Within this low percentage of integration, about ___% of founders will have integrated into an area of the genome that is not transcriptionally active and therefore the transgene is not expressed.
1-5%
15%
Which background mouse is expected to be the least effective in producing a germline chimera?
OUTBRED (will have less donor, more recipient)
Success inbred > F1 > outbred
Male mice are treated with ___ to make them sterile prior to SSC (Spermatogonial Stem Cell) transplant.
busulfan
Which mice and rats have been traditionally used for production of ES cells and recipients of ES cells?
Production: 129 mice, Dark Agouti (DA) rats
Recipients: C57BL/6J mice, F344 rats
What coinfection was thought to play a role in the Ebola-Reston outbreak in cynos in 1989?
SHFV (Arteriviridae)
Facility space should be ___% of the total research space in a building.
15%
What are the four types of circulation patterns in facilities?
- Single-corridor bidirectional pattern
- Single-corridor unidirectional pattern
- Dual-corridor pattern with relatively large animal holding rooms
- Dual-corridor pattern with relatively small animal holding rooms.
Common size for animal holding room in ft and capacity for mouse cages. Each cage will take up approximately ___ft2.
11 ft x 22 ft
Common room space (ft2) and # of cages for a barrier rodent housing room. Approximately how much room does a cage take up?
500-800 cages
250-400 ft2
0.5 ft2
Common room configurations for rodent caging
- Library-style - most efficient
- Internal room corridor style - easiest to service
- Carousel-style - high density and easy to service
What is a common size for a cubicle with 1 rack and with 2 racks? What is the air pressure in the cubicles usually with respect to the service aisle?
4 ft x 6 ft (one rack)
7 ft x 7 ft (2 racks)
Cubicles negative/service aisle positive
Room floor should be sloped @ min ____ of an inch per ft from crown in center of room to floor trough on each side of room. Bottom of trough should slope min ___ of an inch per ft toward a 6 in diam drain w/ trap flush.
3/16th
1/4th
Ratio of support space to animal housing space varies from 30:70 to 70:30. Smaller facilities have (higher/lower) % support space.
higher
Most commonly there is one procedure room for every ___ animal holding rooms
4-8
Options for volatile chemicals that provide 1) personnel protection 2) personnel + product protection 3) altered housing
1) Chemical fume hood
2) Class II Type B2 BSC (100% exhaust)
3) Increased ACH in caging (>50) w/ directional air flow
Allowing radioactive carcasses and waste to decay for __ half-lives permits disposal into nonradioactive waste streams
10
Downdraft tables/workstations should provide ___ capture inches above the work surface
12
What is a commonly used radioisotope for imaging and what is the half life?
F18 (t1/2 = 110 min)
What are the definitions of cleaning, disinfection, and sanitation?
Cleaning = removal of adherent organic and nonorganic debris
Disinfection = decrease in microorganisms
Sanitation = cleaning + disinfection
Drains should be minimum ___ inches, and ___ inches for NHPs/dogs/ag spp
4
6
Exposure time for mechanical washer
1sec @ 180°F
15sec @ 161°F
30 min @ 143°F
Autoclave temp
121-132°C (250-270°F)
Most common type of HVAC and how it works
Single-duct, constant volume reheat (CVR)
Draw in 100% fresh air –> filter, heat/cool, humidify –> deliver to individual room @ constant temp and RH, then reheated to desired temp of the rm level based on room thermostat or building automation system/building management system (BAS/BMS)
What is second most common HVAC system?
What is the benefit of it? How does it work?
Variable air volume (VAV) systems with reheat
More efficient because can tailor ventilation rates (but not much energy savings because already operating at low 10 ACH)
ACH are increased above the minimum set
point depending on the cooling or heat load
What are 3 mechanisms by which HEPA filters trap particles?
Interception, impaction, diffusion
How to create positive or negative pressure spaces? What should the static pressure differential be in terms of inches of water column?
Positive pressure: supply more air to the space than is exhausted; negative, vice versa
between 0.02 and 0.08” of water column (WC). Can go higher for hazardous areas or quarantine, but over 0.25” you can’t open the door
What are the two methods to maintain differential air pressure?
- Volumetric flow tracking: BAS (Building Automation System) adjusts terminal devices to maintain set differential in supply and exhaust air VOLUMES
- Direct pressure control: PRESSURE SENSORS in space provide data to BAS which subsequently adjusts terminal devices
According to the NIH design policies, air velocities should not exceed ___ m/s at an elevation of ___ m, especially when not using filter tops.
0.25 m/s
1.8 meter
How are humidification and dehumidification achieved?
Humidification: injecting steam or atomizing water into supply air stream
Dehumidification: lowering temp of supply air as it masses over cooling coil🡪 condensation🡪 air reheated as supplied to room will contain less moisture and lower RH
Are zone or room humidification systems preferred?
Zone (individual room not really necessary and maintenance-intensive)
Which NHPs needs higher relative humidity?
New world (>50%)
M. fascicularis
(adapted to tropical environments)
3 mechanisms to control light periodicity. Which is the most likely to malfunction?
- Mechanical/electronic timer 2. 1 dedicated timer/room or 3. BAS/BMS
MECHANICAL
How does continuous lighting affect reproduction?
Overstimulation –> development of excess ovarian follicles and permanent vaginal cornification but stop cycling
Two types of light that can be used during dark period with rodents
- Red lamps (>650 nm)
- Sodium lamps (589-589.6nm)
It is recommended to design and operate rodent holding at or below noise criteria (NC)-___. NIH design requirements manual for biomedical lab: not exceed NC-____
NC-40
NC-45
What are the critical macroenvironment relative humidity thresholds for pine shavings and corncob? What does this mean?
A RH above which ammonia accumulates significantly before 7 days in static caging
Pine shavings: 50%
Corncob: 70%
Above these thresholds, NH3 concentrations may exceed, by as much as 14-fold, limits (25 ppm 8-h TWA) established
by the American Conference of Governmental Industrial
Hygienists for human exposure in the workplace.
Of stainless steel 304 vs 316 one of them has ___ added for corrosion resistance.
316 molybdenum
Use of galvanized steel increases risk of ___ toxicity causing ___ and ___.
Zinc; alopecia and achromotrichia
Ferrets, NHPs, rabbits very sensitive!
Which plastic is commonly used for disposable rodent caging and cannot be autoclaved?
Polyethylene terephthalate (PET)
Which three plastics can contain BPA?
Concern for BPA with polycarbonate (PC), polyphthalate carbonate (PPC), and polysulfone (PS)
Generally leach only with high temps but polycarbonate can leach at room temp!
Rodent caging listed from most to least durable
Polysulfone (PS) > polyetherimide (PEI) > polycarbonate (PC) > polypropylene (PP) > polystyrene (PS)
Pneumonic - See Everyone’s Caging PleaSe
According to AWA, for dogs and cats wire flooring must be > __” diameter OR coated with ___ or ___.
1/8
plastic or fiberglass
2 main types of IVCs. Can they be positive and/or negative pressure?
(1) intracage supply/perimeter capture
= positive pressure only
(2) intracage supply/intracage exhaust systems - direct, indirect, or combination
= positive or negative
What are the three types of DIRECT intracage supply/intracage exhaust IVCs?
Air can come in through plenum under cage, bottom of cage, or lid of cage. If air comes in through lid, it goes out through plenum under cage. For other options, air goes out lid.
Where is air supplied and exhausted from for INDIRECT intracage supply/intracage exhaust IVCs? How is it different from the COMBINATION intracage supply/intracage exhaust?
Cage lid
Combination has valve that is activated when cage on rack, air in and flows out via pos pressure
BB authors recommend that ventilation rates be established in IVCs
so that, prior to cage change, MiE NH3 and CO2 are <___ and ___ ppm, respectively, and intracage air flow should be < ___ linear feet per minute (lfpm).
25, 5000
50
MAD units designed for animal research are typically of the Class ___ type, which is defined by Federal Standard No. 209E as HEPA air w/ no more than ___ particles (≥ ___ μm)/cubic foot air
100
100 particles > 0.5 micron
What is the standard for BSCs? How often should they be certified?
NSF/ANSI 49
Certified annually
What are two main types of terminal supply devices for HVAC system?
- Use airflow sensor to adjust to maintain constant volume of air
- Venturi valves (= Phoenix valves) to maintain a pressure differential
What is most common source of gamma irradiation for food? It decreases bacterial/fungal load to ___ bacteria or fungi/g of feed
cobalt source
100
Soft wood bedding (increases/decreases) activity of CYP enzymes
INCREASES
Metabolize drugs faster
Which bedding is excellent at limiting ammonia buildup in static caging?
Corncob
3 types of water purification. What should be added to remove low molecular weight compounds? What is added to RO for disinfection?
reverse osmosis, deionization, and ultrafiltration
Activated carbon
UV (mercury lamp 254 nm bacteriocidal), ozone, autoclave
Water is acidified to pH of ___ to ___ or chlorinated to ___ ppm in order to eliminate ___. Best to add chlorine to ___ water.
2.5-3
6-8
Pseudomonas
RO water (less risk of organic material –> trihalomethanes eg chloroform; less biofilm which oxidized chlorine)
Water pressure for automatic watering for rodents
3 psi
As temp decreases, the microbicidal effect of halogens ___.
decreases
In tunnel washer chemicals are maintained at steady state concentration by measuring ___.
conductivity using conductivity probe (increased detergent concentration –> increased conductivity aka decreased electrical resistance)
What are the two types of budgets for animal care programs?
1) Operating budget - all revenue and expenses for a single fiscal year
2) Capital budget - large expenses that are amortized (buildings, land) or depreciated (equipment) so the acquisition cost is spread over more than one fiscal year
Federal agencies such as the USDA are required to publish any new regulations or changes in existing regulations in the Federal Register and allow a ____-day period for public comments
60
Administration of radionuclides to animals mandates adherence to ___ standards
NRC (Nuclear Regulatory Commission)
Room-level exclusion require more extensive ___ while cage-level requires ___.
PPE
positive-pressure ventilation
The importance of ___ to captive primates is underscored by the observation that it is frequently the most-used enrichment in a variety of ‘nonnutritive’/nonsocial enrichments.
perches
In free-ranging conditions, ____ of non-human primates can occupy from 7-65% of the diurnal activity budget, and has been described as the “single most time-consuming behavior” during some seasons of the year.
foraging
In poultry, factors that influence the development and incidence of feather pecking and cannibalism include light intensity, housing system, genetics, group size, and type of availability of food. Notably, food deficiencies can be a trigger, with dietary ___ playing a particularly important role in feather pecking, and those that do it often ingest the feathers.
FIBER
Which species does belly-nosing as a stereotypy?
Piglets weaned too early; spend less time at feeder and have poor growth rates
What are the definitions of 1) deontological ethical theory, 2) teleological ethical theory, 3) and utilitarianism
1) right and wrong
2) consequence
3) more benefit to the most people with less harm to least people (a type of #2)
At high densities, rats have a __________ hierarchy
despotic (one being rules)
Rats with play with _________ but not _____________.
each other
objects
Each hamster burrow consists of ___(#) tunnels off of the nest chamber
two ; a toilet chamber and a food storage chamber
Gerbils are a ______ (social/solitary) species that build complex burrow systems. When in captivity, they exhibit stereotypic ______ behavior – therefore, it is important to provide substrate to allow for burrowing or provide _______ chambers.
Social
digging
L-shaped
Guinea pigs are _____ (social/solitary) animals that have a wide vocal repertoire. They have _____ (number) of distinct calls. Their stereotypies include chewing behaviors, which can include on hair and ears – but the provision of ______ decreases hair loss.
social
11
hay
The single most important factor in the development of abnormal behaviors of NHPs is: ________
nursery rearing
Implantation of tumor material into the ____ requires special justification due to impaired mobility and increased pain.
muscle
What does CIOMS stand for? When were the CIOMS Principles created and what is their full name?
Council for International Organizations of Medical Sciences, 1985, International Guiding Principles for Biomedical Research Involving Animals
What does IRAC stand for and what is the full name of the IRAC Principles?
US Interagency Research Animal Committee, US Government Principles for the Utilization and Care of Vertebrate Animals Used in Testing, Research and Training
(based on CIOMS principles and in the PHS Policy)
