Chapter 3. Biology and Diseases of Mice Flashcards
1
Q
What is the scientific classification of the laboratory mouse?
A
Order Rodentia Superfamily Muroidea Family Muridae Subfamily Murinae Genus Mus Clade M. musculus, collectively called the 'house mouse'
2
Q
What is the native range of the genus Mus?
A
Eurasia and North Africa
3
Q
What is an ‘aboriginal’ species?
A
Species that lives independent of humans.
Species of the genus Mus have been classified as either aboriginal or commensal
4
Q
What is a ‘commensal’ species?
A
Species that have coevolved and geographically radiated with human civilization since the dawn of agriculture 12,000 years before present (bp).
(Species of the genus Mus have been classified as either aboriginal or commensal)
5
Q
What is the origin of the genus name “Mus”?
A
From Sanskrit, Mush: to steal.
6
Q
Where did the progenitor of the M. musculus clade arise?
A
Northern Indian subcontinent. Diverged into genetically isolated and distinct species or subspecies due to geographic barriers (mountain ranges). There is debate over whether the taxa in this clade are species or subspecies, and some have referred to them an ‘incipient species’, but their genetic divergence is blurring as they colonize the world and hybridize.
7
Q
What is an incipient species?
A
A group of a particular species that are about to become genetically isolated from the rest of the species, though at this time can still reproduce with other groups of the species before their gene pools become too distinct.
8
Q
What is the approximate percentage make-up of different M.m. species in the laboratory mouse genome?
A
~60% M.m. domesticus
~30% M.m. musculus
~10% M.m. castaneus
9
Q
Where did the M.m. musculus and M.m. castaneus contributions to the laboratory mouse genome originate from?
A
Primarily derived from M.m molossinus Japanese fancy mice.
10
Q
M.m. domesticus
A
Indigenous to western Europe and southwest Asia.
11
Q
M.m. musculus
A
Indigenous to eastern Europe and northern Asia.
12
Q
M.m. castaneus
A
Indigenous to southeast Asia.
13
Q
M.m. molossinus
A
Indigenous to Japan and the Korean peninsula.
14
Q
Describe the history of Asian ‘fancy mice’.
A
The cohabitation of humans with commensal mice gave rise to captive breeding for coat color and behavioral variants in China over 3000 years ago. By the 1700s, mouse ‘fanciers’ in Asia had created many varieties of fancy mice, as did European fanciers, who subsequently acquired Asian stocks, esp. Japanese M.m. molossinus to mix with European M.m. domesticus.
15
Q
Most laboratory mouse strains are closely genetically related, since many were derived from a genetically mixed but small number of fancy mice from which singly mouse breeder at the beginning of the 20th century?
A
Abbie Lathrop’s Granby Mouse Farm, Massachusetts
16
Q
Most inbred lab mice share a common maternal mitochondrial genome derived from _______ and a common Y chromosome contributed by _______.
A
Maternal mitochondrial genome = M.m. domesticus.
Y chromosome = M.m. musculus, through its contribution to the genome of M.m. molossinus.
17
Q
C57BL/6 mice contain minor genetic elements from what mouse species?
A
M. spretus.
18
Q
Name wild aboriginal species of mice that are not members of the M. musculus clade, but have been used to establish inbred lines of mice.
A
M. spretus.
M. caroli
19
Q
Abbreviation 129: A
A
A strains
20
Q
Abbreviation 129: AK
A
AKR strains
21
Q
Abbreviation 129: B
A
C57BL strains
22
Q
Abbreviation 129: B6
A
C57BL/6 substrains
23
Q
Abbreviation 129: B10
A
C57BL/10 substrains
24
Q
Abbreviation 129: BR
A
C57BR strains
25
Abbreviation 129: C
BALB/c strains
26
Abbreviation 129: C3
C3H strains
27
Abbreviation 129: CB
CBA strains
28
Abbreviation 129: D
DBA strains
29
Abbreviation 129: D1
DBA/1 substrains
30
Abbreviation 129: D2
DBA/2 substrains
31
Abbreviation 129: HR
HRS strains
32
Abbreviation 129: L
C57L strains
33
Abbreviation 129: R3
RIIIS strains
34
Abbreviation 129: J
SJL strains
35
Abbreviation 129: SW
SWR strains
36
What were the first autosomal linked mouse genes found to be linked in 1915?
Albino and pink-eyed dilution
37
How many pairs of chromosomes do mice have?
20 telocentric chromosomes in their haploid genome (40 chromosomes in all).
Differentiated by size and patterns of transverse bands. Designated by Arabic numbers in order of decreasing size.
In the 1970s, chromosome rearrangements were used to assign known genetic linkage groups - designated by Roman numerals - to specific chromosomes and for determining locus order with respect to the centromere.
38
What lab technique can be used to physically locate genes on chromosomes?
Fluorescent in situ hybridization (FISH)
39
Quantitative trait loci (QTL)
Chromosomal regions containing genes that influence a quantitative trait.
This methodology has allowed for mapping genes and identifying similarities between mouse and human genomes.
40
Histocompatibility complex
One of the most thoroughly studied genetic systems of the mouse.
Histocompatibility (H) loci control expression of cell surface molecules that modulate critical immune responses.
41
On what chromosome is the major histocompatibility complex (MHC) located in mice?
Chromosome 17.
42
H2 complex
Mouse major histocompatibility complex genes are arranged in a complex gene cluster - the H2 complex, coding for proteins either directly performing or associated with antigen presentation to T lymphocytes.
Several loci, including K, D, L, I-A, I-E.
Minor H2 loci groups are scattered throughout the genome and are responsible for delayed graft rejection.
Genes associated with the H2 complex also control other immunological functions - cell-cell interactions in primary immune responses, level of response to a given antigen, immune-mediated responses to infectious agents such as viruses and complement activity.
43
H2 haplotypes
Inbred strains of mice, being homozygous, each have unique sets of H2 alleles, termed H2 haplotypes.
BALB H2 haplotype = H2^d
C57BL H2 haplotype = H2^b
44
International ImMunoGeneTics (IMGT) Information System
Provides details on H2 haplotypes for various inbred mice.
45
International Mouse Genome Sequencing Consortium
Developed a robust physical map and high-quality genome sequence of the C57BL/6J mouse in 2002.
46
Mouse Genomes Project/Wellcome Trust Sanger Institute
Extending mouse genome sequencing effort to include the genome sequences of 17 key mouse strains.
47
European Nucleotide Archive
Completed and evolving mouse sequence data is available through this archive.
48
Mouse Genome Informatics database
Inbred mouse strains, natural mutants, induced mutants, transgenic lines, and targeted mutant lines of mice are catalogued.
49
Mutant Mouse Regional Resource Centers
In the US, there are four regional NIH-supported centers, which link to international mouse repositories in Europe, Japan, Canada, China, and Australia, as well as additional resources programs in the US through the International Mouse Strain Resource.
50
International Mouse Strain Resource (IMSR)
For depositing, archiving, and distributing mutant mouse and embryonic stem cell lines to the scientific community.
51
Which groups are working on three major targeted gene knock-out programs, all utilizing C57BL/6N embryonic stem cells?
Funded by the NIH, the European Community, and Genome Canada.
Knock Out Mouse Project (KOMP), the European Conditional Mouse Mutagenesis Program (EUCOMM), and the North American Conditional Mouse Mutagenesis Project (NorCOMM).
52
What are the three distribution centers for the targeted knock-out programs with C57BL/6N embryonic stem cells?
Mouse lines will be available through:
German Resource Center for Genome Research (RZPD), the KOMP repository, and the Canadian Mouse Consortium (CMC).
The repositories are all linked to IMSR, and provide access to mice, germplasm, genomic detail, and phenotypic data.
53
International Mouse Phenotyping Consortium (IMPC) and the Mouse Genome Database (MGD)
Genetic, genomic, and biological data on mice are available through these groups.
54
C.C. Little
Developed the first inbred mouse strain (DBA) in 1909
55
Strains
Term for inbred mouse strains
| Achieved by 20 or more brother x sister (filial) generations.
56
Isogenic OR Syngenic
Genetically identical
| Mice within an inbred strain are isogenic or syngenic to other mice of the same strain and sex.
57
When is a mouse strain FULLY inbred?
Because of residual heterozygosity, a strain is not fully inbred until after 60 filial (F) generations.
Most commonly used inbred strains represent 200 or more F generations.
58
Substrain
When branches of an inbred strain are separated, spontaneous mutations, residual heterozygosity, and retroelement integrations results in genetic differences.
Branches of an inbred strain become SUBSTRAINS if:
1) They are separated before F40
2) If branches have separated for 100 generations
3) If genetic differences arise
59
Recombinant inbred
RI strains are sets of inbred strains of mice derived from crossing two inbred strains, and developed by single pair random matings of sibling mice from the F2 generation, thereby creating separate breeding lines.
When two inbred strains are crossed, the F1 hybrids are genetically identical (isogenic) but maximally heterozygous (with chromosomes of each chromosomal pair separately contributed by each parent strain). F2 hybrids are maximally genetically diverse from one another (with chromosomes of both chromosomal pairs containing a mixture of contributions from each parental strain). With subsequent F generations, mice once again approach inbred status. This technique is used for creating recombinant inbred (RI) strains.
60
Describe the separate breeding lines for recombinant inbred strains.
Each line created is maintained separately, and then propagated by brother-sister matings for 20 generations, with each line becoming a separate inbred strain, but belonging to a set of recombinant inbred strains.
Recombinant inbred sets are generally limited to two parental strains.
61
What is the benefit of using recombinant inbred strains?
Recombinant inbred mice are useful for mapping phenotypic or quantitative traits that differ between the progenitor strains.
62
Collaborative Cross mice
An international effort to increase allele diversity among recombinant inbred strains by creating the Collaborative Cross - a panel of recombinant inbred strains being generated by mixing the genomes of eight disparately related inbred strains.
63
Which 8 mouse strains are part of the Collaborative Cross?
A/J, C57BL/6J, 129S1/SvImJ, nonobese diabetic (NOD)/ShiLtJ, NZO/HILtJ, CAST/EiJ, PWK/PhJ, and WSB/EiJ.
These 8 strains capture nearly 90% of the known genetic variation present among laboratory mice.
64
Recombinant congenic strain
Sets of inbred strains derived in a manner similar to that for recombinant inbred strains, except that one or more backcrosses to one parental strain (designated the background strain) are made after the F1 generation, before inbreeding has begun.
The other parental strain is designated the donor strain.
65
What determines the proportion of background strain and donor strain genomes in a recombinant congenic strain?
The number of backcrosses proceeding inbreeding.
66
Advanced intercross lines (AIL)
A type of recombinant inbred line. Made by producing an F2 generation between two inbred strains and then, in each subsequent generation, intercrossing mice but avoiding sibling matings. This increases the possibility of tightly linked genes recombining.
67
What is the purpose of advanced intercross lines?
To increase the possibility of recombination between tightly linked genes.
68
Co-isogenic
When a mutation arises spontaneously or is induced within an inbred strain, that mutant mouse becomes co-isogenic with the parental inbred strain, being virtually identical except for the single mutant allele.
69
Backcrossing
If a mutation that arose in one inbred strain is desired within the background of another inbred strain, this is accomplished by backcrossing - an F1 hybrid is created by mating the donor mutant strain to the desired background strain, with subsequent matings to the background strain while retaining the mutant locus.
70
Congenic
Strains that are genetically identical except for one mutant locus.
An F1 hybrid is created by mating the donor mutant strain to the desired background strain, with subsequent matings to the background strain while retaining the mutant locus. After 10 backcross generations, the mutant mouse line is congenic to the background inbred strain.
71
Are congenic mice ever co-isogenic?
No, as the preserved mutant locus in a congenic mouse is invariably surrounded by flanking DNA, which may significantly influence phenotype.
72
Stock
The name for outbred mice.
Defined as a closed population (for at least four generations) of genetically variable mice that are bred to maintain maximum heterozygosity.
73
Outbreeding
Can only be achieved with a large breeding population using a systematic breeding scheme, or randomized selection of breeders from the population.
Ex: in a population of 25 breeding pairs, heterozygosity will decrease at 1% per generation with standard randomization techniques.
74
Circular pair mating system
An outbreeding scheme in which each pair is mated only once. Cages are visualized in a circle, and each cage contains one breeding pair in the nth generation. Another 'circular' set of cages serves as the breeding nucleus for the n+1 generation. Each mated pair in the nth generation contributes one female and one male to the n+1 generation. Outbreeding is accomplished by assigning the female and male derived from each nth generation cage to different cages in the n+1 generation.
75
Swiss origin mice
Most outbred stocks are of 'Swiss' origin, derived from nine mice imported to the US in 1926. Although considered outbred, they have a high degree of homozygosity, exemplified by the fact that many Swiss mouse stocks are blind due to the homozygous recessive rd1 allele.
76
Why are many Swiss stocks blind?
Homozygous recessive rd1 allele
77
Diversity Outbred mouse
A hetergeneous stock derived from the same eight founder inbred strains of the Collaborative Cross.
78
Consomic
Also known a chromosome substitution strains.
| Inbred mice that are congenic for entire chromosomes, and are useful for studying polygenic traits.
79
Conplastic
Inbred mice that are congenic for different mitochondrial genomes (mtDNA) contributed by other inbred strains, other subspecies, or other species of Mus.
80
Recombinant inbred
When two inbred strains are crossed, the F1 hybrids are genetically identical (with chromosomes of each chromosomal pair separately contributed by each parent strain) but maximally heterozygous
81
How do you perpetuate breeding of segregated inbred strains?
Brother-sister mating system for more than 20 generations with heterozygosity for the mutations forced by 1) backcrossing, 2) intercrossing, 3) crossing and intercrossing, 4) backcrossing and intercrossing.
82
How do you perpetuate breeding of a coisogenic inbred strain?
Perpetuate the mutation by 1) brother-sister mating within the strain of origin, 2) backcross or cross-intercross system with strain of origin as parent strain, 3) brother-sister mating with heterozygosity forced by back- or intercrosses, 4) brother-sister mating between homozygotes.
83
How do you perpetuate breeding of a congenic inbred strain?
Perpetuate the transferred mutation by 1)backcross or cross-intercross system with strain of origin as parent strain, 2) brother-sister mating with heterozygosity forced by back- or intercrosses, 3) brother-sister mating between homozygotes.
4) Brother-sister mating within strain of origin may be used after 10-12 generations of backcrossing with periodic backcrosses to background strain
84
Radiation mutagenesis
Radiation was one of the earlier methods for in vivo mutagenesis, but in vitro radiation of embryonic stem (ES) cells is also performed
85
Chemical mutagenesis
Involves in vivo treatment of male mice or in vitro treatment of ES cells with mutagenic chemicals such as ethylmethanesulphonate (EMS) or N-ethyl-N-nitrosurea (ENU), which induce point mutations in DNA.
86
Transgenic mouse
Technically any mouse in which foreign DNA has been integrated into its genome, regardless of method.
Commonly refers to mice that are genetically altered by additive transgenesis.
87
Additive transgenesis
Microinjection of foreign DNA into the pronucleus of a fertilized egg. Each ensuing embryo results in a genetically different "founder" mouse, since the transgene is integrated in random sites of the genome of each founder mouse.
88
Hemizygous
Since injected DNA is not homologous to the mouse genome and is not an allele, transgenic founder mice are hemizygous (rather than heterozygous) for the transgene until the mice carrying the transgene are bred into homozygosity for the transgene.
89
Transgene
A gene that has been extracted from the DNA of one organism and transferred into the DNA of an organism of another species.
Transgenes typically integrate at tandem repeats, copy numbers affect phenotype of each founder, and may be lost in subsequent generations, thereby changing the phenotype of the mouse line.
90
Promoter
Transgenes are often constructed with an upstream promoter, which confers widespread (ubiquitous) or tissue-specific expression of the cDNA, so that the transgene expression pattern reflects the expression pattern of the promoter.
91
Tetracycline (tet) regulatory system
Treatment of mice with tetracycline or doxycycline induces up- or down-regulation of the transgene.
92
Inducible
Transcriptional regulation of a transgene is under drug-dependent regulatory control.
93
Gene trapping
High-throughput approach that randomly introduces insertional mutations within the genome. Vectors contain a gene trapping cassette with a promoter-less reported gene and/or selectable genetic marker flanked by an upstream 3' splice site and a downstream termination sequence.When inserted into an intron of an expressed gene, the gene trap is transcribed from the endogenous promoter of that gene. Gene traps simultaneously inactivate and report the expression of the trapped gene at the insertion site, and provide a DNA tag for the rapid identification of the disrupted gene.
94
Targeted mutation
Achieved by homologous recombination of specific sites within the genome of ES cells. Homologous sequences flank the upstream and downstream regions of the targeted gene, and the construct between the flanking sequences may inactivate (knock out) or replace (knock in) a gene, and typically contains a reporter gene to track the integration.
95
Cre
Site specific DNA recombinase from the coliphage P1; cuts at loxP sites; can be made tissue specific using specific promoter.
96
Outcome of Cre if loxP sites are oriented in opposite directions?
Cre recombinase will mediate the inversion of the floxed segment.
97
Outcome of Cre if loxP site are on different chromosomes (trans)?
Cre will mediate a chromosomal translocation.
98
Outcome of Cre if loxP sites are oriented in the same direction of the same chromosome (cis)?
Cre will mediate deletion of the floxed segment.
99
Describe how Cre recombinase functions in a mouse model.
The floxed mutation is created in ES cells, and the transformed ES cells are developed into a mouse with the conditional mutation. The conditional mutation mouse is then genetically crossed with a Cre transgenic mouse, in which Cre recombinase is under the control of a ubiquitous or tissue specific promoter. Wherever and whenever Cre is expressed, Cre recombinase will recognize and recombine the loxP sites.
100
FLP
Site specific recombinase from Saccharomyces cerevisiae.
101
ES cells are pluripotent with the full genetic capacity to develop into mice when implanted into the ________ of a developing embryo.
Blastocyst.
102
What caused genetic corruption of the 129 mouse in the 1970-1980s?
Interest in embryonal carcinomas (teratomas) that arose in relatively high frequency in the testes of 129 mice. Early gene transfer experiments in the 70s and 80s led to the development of ES cell lines derived from several different 129 strains. This early emphasis on teratomas prompted creation of 'better' 129 mouse lines that were more prone to developing testicular teratomas. This gave rise to the need to revise 129 mouse nomenclature.
103
What are the most commonly used mouse ES cells for homologous recombination? What mouse ES cells are recent international knockout mouse programs using?
1) 129
| 2) C57/6N
104
Chimera
Transformed ES cells are microinjected into the inner cell mass of recipient blastocysts, which are then implanted into the uteri of pseudopregnant surrogate mothers. The pups that are born are composed of a mixture of cells derived from recipient blastocysts and the transformed ES cells = chimera.
105
Why are most ES cell lines XY?
For chimera production, the goal is for male chimeric progreny to produce spermatozoa of ES cell origin (containing the mutation) in order to create F1 progeny by mating the chimera with the desired background strain. XY ES cells lines favor 129 male chimerism.
106
If a chimeric male created with C57BL/6N ES cells is bred directly with C57BL/6 mice, what type of line is created?
Co-isogenic.
107
If a chimeric male created with 129 ES cells is bred directly with C57BL/6 mice, what type of line is created?
Congenic.
108
Aggregation chimera
An alternative to traditional chimeric production. Allows ES cells to aggregate with a developing embryo to form blastocysts in culture, then the chimeric blastocysts are implanted.
109
dsRNA
Double-stranded RNA.
110
RNAi
RNA interference. Functions through short dsRNA. Used to produce transgenic "knock down" mice.
The ds RNA is enzymatically processed into small molecules, termed small interfering RNA (siRNA), which find homologous target mRNAs, resulting in interference.
Constructs are introduced into mouse ES cells by electroporation and lentiviral infection.
Although RNAi knock down mice are genetically stable, RNAi-mediated transgenesis is never complete, has variable tissue expression, and cannot induce point mutations.
111
What is thought to be the evolutionary basis for why RNAi works?
Believed to be a self-defense mechanism against viral infection.
112
shRNA
Small hairpin RNA. To adapt the RNAi phenomenon for the generation of transgenic mice, shRNA can be expressed in the same way as other transgenes in mice, resulting in processing of the shRNA into siRNA with gene-silencing effects.
113
Zinc finger nucleases (ZFNs)
Comprised of 3 to 6 tandem zinc finger proteins, each of which targets a specific 3 bp nucleotide sequence. Paired ZFNs are generated, with each half of the pair targeting opposite DNA strands, allowing dimerization of the nonspecific endonuclease, Fok1, which is required for introduction of double-stranded breaks in the DNA of interest.
114
Fok1
Nonspecific endonuclease. Used in engineered endonuclease technology with ZFNs and TALENs.
115
Transcription activator-like effector nucleases (TALENs)
Composed of tandem repeats of 33-35 amino acids, each with nucleotide specificity occurring in two hypervariable amino acids, the 'repeat variable di-residue (RVD)', at positions 12 and 13.
116
Clustered regularly interspersed short palindromic repeats (CRISPR)
Paired with CRISPR-associated (CRISPR/Cas) systems, are RNA-guided endonuclease systems that target specific DNA sequences.
117
Cas
Cas proteins, rather than Fok1, produce double-stranded breaks in DNA with CRISPR.
118
How are double stranded breaks generated by engineered endonuclease technology repaired by host cells?
By nonhomologous end joining (NHEJ) or, less commonly, homologous recombination.
119
Nonhomologous end joining (NHEJ)
An error-prone mechanism of double-stranded DNA break repair in eukaryotic genomes in which damaged nucleotides are removed and blunt ends of strands are joined. Results in insertions or deletions (indels) with a relatively high frequency, which can result in gene disruption.
120
Homologous recombination (HR)
A repair pathway for double strand breaks involving the undamaged sister chromatid, which provides a template for high fidelity repair. (BRCA1 and BRCA2 involved in this)
Certain manipulations of engineered nucleases can increase HR efficiency - e.g., nucleases can be engineered to generate a single strand break, rather than a double strand break, and the resulting nickases increase the incidence of HR with high fidelity.
HR allows for knock-ins, specific point mutations, or for the generation of larger modifications such as insertions of loxP sites.
121
How can vectors encoding engineered endonucleases be introduced into mice?
1) Injected into mouse embryos by pronuclear injection of DNA.
2) Intracytoplasmic injection of RNA.
3) Transfection of mouse ES cells.
122
What are some advantages of engineered endonuclease (EE) technologies over more traditional transgenic methods?
1) Ability to target DNA and induce mutations in any background strain of mouse negating the need to backcross to the desired strain.
2) Multiple genes can be targeted with CRISPRs simultaneously, thus avoiding the need to cross single knockout animals.
3) It is possible to obtain bi-allelic mutations in some cases, allowing for the generation of functional gene knockout animals in a single generation.
4) CRISPRs tend to be particularly cost-effective and easy to design, with minimal restrictions for targeting specific DNA sequences.
123
The International Committee on Standardized Genetic Nomenclature for Mice and Rats
Established in the 1950s; responsible for genetic nomenclature rules.
124
Nomenclature for substrains
Appending a forward slash (/) after the inbred strain name. Ex: C57BL/6.
Substrains can be cumulative (contain multiple laboratory codes)
125
International Laboratory Code Registry
Maintained at ILAR. Substrains of inbred strains are designated using standardized laboratory codes.
Lab codes are 1-5 letters, starting with one uppercase letter and then lowercase letters
126
Nomenclature for F1 hybrid
Female partner is listed first. R1 strain sets that are derived from two parental inbred strains are identified by an 'X' between the two parental strains followed by a hyphen designating the specific R1 line.
127
Nomenclature for mutant gene and allele
Parent gene is in italics, first letter capitalized. The mutant allele is designated in superscript. Ex: Lyst^bgJ
128
Nomenclature for transgenic strain
Strain and substrain name, followed by "Tg(YYY)#Zzz, where Tg stands for transgenic, YYY defines the transgene as a brief description of the inserted DNA, # is the assigned number in the series of events generated using a given construct, and Zzz is the Lab Code. Ex: FVB/N-Tg(MMTV-erb2)1Led
129
Nomenclature for when a transgene causes an insertional mutation
The mutant allele of the gene is designated using the gene symbol and an abbreviation for the transgene as a superscript. Ex: -Abc^tg1Zzz
130
Nomenclature for a targeted mutation
'tm' for targeted mutation. Ex: Cftr^tm1Unc
131
Nomenclature for congenic
When the backcross generation is at N10 or more, the '.' symbol is used between the background inbred strain and the donor strain. Ex: C57BL/6N.129P2/OlaHsd-Abc^tm1Zzz
132
Nomenclature for incipient congenic
When backcrossing is incomplete but at the N5-N9 generation, the mouse is an incipient congenic, designated with a ';'. Ex: B6;129-Abc^tm1Zzz
133
Nomenclature for donor strain of mixed origin and outbred
If the background strain is mixed genetic origin, it is designated STOCK. If the donor strain is mixed origin, it is designated Cg. Outbred stock that meets specific criteria is designated by placing the Lab Code before the stock symbol, separated by ':'. Ex: Hsd:ICR is an ICR(Swiss) outbred stock maintained by Harlan Sprague Dawley.
134
Why do some mouse vendors house production colonies in open top caging?
To expedite detection of pathogen transmission should a break occur.
135
Rank the common plastics used to make static shoebox mouse caging in order of decreasing cost and durability.
Polycarbonate, polypropylene, polystyrene.
136
Why are solid bottom cages preferred for mice?
Injury risk to limbs and thermoregulation issues for neonates and hairless mice housed on wire-mesh floors.
137
List potential tests of rodent bedding quality.
Chemical properties: pesticides and polychlorinated compounds, mycotoxins, nitrosamines, detergent residues, ether-extractable substances, heavy metals.
Physical properties: particle uniformity, absorptivity, ammonia evolution, visible trauma and irritant potential.
Microbiological properties: standard plate count, yeasts and molds, coliforms, Salmonella, Pseudomonas.
138
How much do mice eat in a day?
3-5 grams of feed per day after weaning.
139
How much water do mice drink in a day?
Adult mice drink 6-7 mL a day.
140
In what forms may mouse diets be supplied?
Standard
Irradiated - virtually free of live microorganisms but have the risk of residual, radio-resistant bacteria
Autoclaved - higher in heat-labile nutrient content
141
Why should animal care programs avoid using fresh produce, grains, fish meal, or other supplements to feed mice?
To minimize exposure of colonies to pathogens or harmful chemicals such as pesticide residues or phytoestrogens
142
What is the basic recommended nutrient profile for mouse diets?
20-25% protein
5-12% fat
2.5% fiber
45-60% carbohydrates
143
T or F: Inbred mice tend to gain weight faster than outbred mice and are heavier at maturity.
FALSE. Outbred mice tend to gain weight faster and are heavier at maturity.
144
Normal mouse body temperature
98.8-99.3 F (37-37.2 C)
145
Normal mouse heart rate and blood pressure
HR: 310-840 beats/min
Systolic: 133-160 mmHg
Diastolic: 102-110 mmHg
146
Normal mouse respiratory frequency
163/min
147
What is the mouse thermoneutral zone?
~29.6-30.5 C
148
Are mice true endotherms?
No. The mouse has no sweat glands, cannot pant, and its ability to salivate is severely limited. Its primary means of cooling in the wild is behavioral - going into a burrow. In confinement, mice will start to die at 37 C ambient temperature.
Neonatal mice are ectothermic and do not have well-developed temperature control before 20 days of age.
149
What is the mouse response to cold exposure?
Non-shivering thermogenesis via brown fat. A resting mouse acclimated to cold can generate heat equivalent to triple the basal metabolic rate, a change that is greater than for any other animal.
Mice cannot tolerate nocturnal cooling as well as larger animals that have a greater heat sink (should NOT lower facility temps at night).
150
Respiratory anatomy of the mouse
Anterior resp tract: Nostrils, nasal cavity, nasopharnyx
Intermediate tract: Larynx, trachea, bronchi
Posterior tract: Lungs
5 lung lobes: 1 left lobe; right lobes = superior, middle, inferior, and postcaval lobes
151
A mouse at rest uses about 3.5 mL O2/g/h (22 times more than an elephant). What physiologic changes support this high oxygen requirement/metabolic rate?
High alveolar PO2, a rapid resp rate, a short air passage, a moderately high RBC concentration, high RBC hemoglobin and carbonic anhydrase concentrations, a high blood O2 capacity, a slight short in the O2-dissociation curve (enabling O2 to be unloaded in tissue capillaries at a high PO2), a more pronounced Bohr effect
152
Kidneys from males of many inbred strains are consistently ___________ than kidneys from females.
Heavier. In all mice, the right kidney is normally located anterior to the left kidney.
153
Describe the glomeruli of mice.
About 74 um in diameter
About half the size of glomeruli in rats. Mice have 4.8x as many glomeruli as rat, and the filtering surface per gram of tissue is twice that of the rat
154
Why is mouse urine so concentrated?
Mice only excrete a drop of two of urine at a time.
The high concentration is made possible by long loops of Henle and by organization of giant vascular bundles (vasa recta) associated with the loops of Henle in the medulla.
Mice can concentrate to 4300 mOsm/l, whereas humans can concentrate to a max of 1160 mOsm/l.
155
Is proteinuria normal in mice?
Yes - they normally excrete large amounts of protein.
Taurine is always present in mouse urine, tryptophan is always absent.
Creatinine is also excreted in mouse urine (different from other mammals).
Mice excrete much more allantoin than uric acid.
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What is the creatinine/creatine ratio for fasting mice?
~ 1:1.4
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What saliva is produced by the mouse submaxillary salivary gland?
ONLY seromucoid saliva (a mixed gland in most animals)
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Which portions of the mouse stomach are keratinized or glandular?
The proximal portion is keratinized, the distal portion is glandular.
Gastric secretion continues whether or not food is present.
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What anatomical feature of the mouse esophagus assists in oral gavage?
The esophagus is lined by thick cornified epithelium.
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How much bacteria does the ceca of a normal mouse contain?
Up to 10^11 bacteria/g of feces.
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Schaedler Flora
In the mid-1960s, Schaedler was the first to colonize germfree mice with selected bacteria isolated from normal mice.
These defined bacteria include aerobic bacteria and some less oxygen-sensitive anaerobic organisms. The extremely oxygen-sensitive (EOS) fusiform bacteria that make up the majority of the normal microflora of rodents were not included due to technical difficulties with isolation and cultivation.
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Altered Schaedler Flora (ASF)
In 1978, the National Cancer Institute (NCI) revised the Schaedler flora in order to standardize the microbiota used to colonize germfree rodents.
ASF consists of 4 members of the original flora (2 lactobacilli, Bacteroides distasonis, and the EOS fusiform bacteria), a spiral-shaped bacterium, and three new fusiform EOS species.
ASF 361 - Lactobacillus salivarius (16S rRNA sequence is essentially identical to strains of L. murinus and L. animalis - all these strains probably belong to the same species)
ASF 360 - novel lactobacillus that clusters with L. acidophilus and L. lactis
ASF 519 - Parabacteroides sp.
ASF 457 - spiral shaped; Flexistipes phylum (formerly called Mucispirillum schaedleri)
ASF 492 - Eubacterium plexicaudatium
ASF 500 - Firmicutes
ASF 502 & ASF 356 - Clostridium sp.
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Quantification of ASF along the GI tract of mice
Individual strain abundance is dependent on oxygen sensitivity.
Microaerotolerant Lactobacillus murinus ASF361 is present in the upper GI.
Obligate anaerobic ASF strains are predominant in the cecum and colon.
ASF 356, 457, & 492 - highest levels in cecum and colon
ASF 500 - spike in level in esophagus, most common in cecum, level drops somewhat from cecum to colon
ASF 361 - very common in esophagus, common in small intestine and cecum, level dropping off in colon
ASF 502 - most common in cecum, less so in colon
ASF 519 - very common in cecum, most common in colon
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What do ASFs 492, 500, 502, & 356 have in common?
All EOS fusiform bacteria, group phylogenetically with the low-G + C content gram-positive bacteria (Firmicutes-Bacillus-Clostridium group)
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Do mice have tonsils?
No, mice do not have palatine or pharyngeal tonsils.
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Describe the anatomy of the mouse spleen.
Male spleens may be 50% larger than those of females.
Most lymphocytes enter and leave the spleen in the bloodstream. The white pulp is organized along the central arteriole and is subdivided into T- and B-cell zones. The periarteriolar sheath is composed mainly of CD4+ and CD8+ T cells and lymph follicles. The red pulp consists of sinusoids and hemoreticular tissues. Cellular and humoral components of immunity are distributed to the bloodstream and tissues by efferent lymphatic ducts, which empty into the venous system.
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What are the effects of thymectomy in newborn mice?
Maturation and differentiaton of T lymphocytes in the thymus is not complete in newborn mice. Thymectomy results in a decrease of circulating lymphocytes and marked impairment of certain immune responses, particularly cellular immune responses.
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What are the effects of thymectomy in adult mice?
No immediate effects. Several months later mice may develop a progressive decline of circulating lymphocytes and impaired cellular responses.
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What is mucosa-associated lymphoid tissue (MALT)?
Includes all peripheral lymphoid tissues connecting to cavities communicating with the external milieu; includes Peyer's patches, cecal lymphoid tissue, and lymphoid tissue in the upper and lower respiratory tract and the genitourinary system. MALT contains more lymphoid cells and produces greater amounts of immunoglobulin than both the spleen and the lymph nodes.
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Where do nutrients for bone marrow come from?
Reticular tissue rich in blood vessels and adipose cells.
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Define axial skeleton.
Skull, vertebrae, ribs, and sternum,
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Define appendicular skeleton.
Pectoral and pelvic girdles and the paired limbs.
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What is the normal vertebral formula for the mouse?
C7 T13 L6 S4 C28
| Variations among strains are especially seen in the thoracic and lumbar regions.
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What is the normal mouse dentition?
1 incisor & 3 molars in each quadrant.
Develop and erupt in sequence from front to rear. The 3rd molar is the smallest tooth in both jaws; the upper and lower 3rd molar may be missing in wild mice and some inbred strains.
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What are the paired clitoral glands in the mouse?
Homologous to the male preputial glands. Secrete a sebaceous substance through ducts entering the lateral wall of the clitoral fossa.
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How many mammary glands are typically found in the mouse?
Five pairs - 3 cervicothoracic and 2 inguinoabdominal. Are extensive in reach over the cervical, axillary, and inguinoabdominal flank regions.
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Describe the roles of FSH, LH, and prolactin in sexual maturation in the mouse.
FSH - promotes gametogenesis in both sexes.
LH - promotes secretion of estrogen and progesterone in the female and androgen in the male.
Prolactin - promotes lactation and the development of the ovary during pregnancy.
These hormones also ensure proper maintenance of the repro tract and modulate behavior to promote successful mating.
When the cervix and vagina and stimulated physically during estrus, prolactin is released from the anterior pituitary to enable the corpus luteum to secrete progesterone. Secretion occurs for ~13 days. If fertilization has occurred, the placenta takes over progesterone production. If fertilization does not occur, a pseudopregnant period ensures, during which estrus and ovulation do not occur.
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When does the hypophysis play a role in sexual maturation in the mouse?
Usually responsive to hormonal influence in the male by day 6, and in the female by day 12.
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At what age does sexual maturation occur in the mouse?
28-49 days
Female: ovarian follicle development begins at 3 wks of age and matures by 30 days. Rising levels of gonadotropins evoke signs of sexual maturity at about the same age. Estrogen-dependent signs such as cornification of the vaginal epithelium at the vaginal opening can occur as early as 24-28 days.
Male: puberty is slightly later than in females (up to 2 wks).
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Describe the estrous cycle of the mouse.
Polyestrous; cycles every 4-5 days.
1st phase - proestrus
2nd phase - estrus
3rd phase - metestrus
4th phase - diestrus
Active epithelial growth in the genital tract during proestrus and estrus culminates in ovulation. Diestrus is a period of quiescence or slow cell growth.
Spontaneous ovulators. Ovulation may not coincide with estrus, b/c estrus is dependent on gonadal hormones and ovulation is responsive to gonadotropin. Cyclicity of estrus and ovulation in controlled by the diurnal rhythm of the photoperiod.
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Define the Whitten Effect.
Estrus may be suppressed in group-housed female mice and reentry into estrus can be synchronized by exposure to pheromones in male mouse urine.
(Once exposed to urine, female mice will be in estrus within 3 days, with a second estrus in about 11 days.)
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Define the Bruce Effect.
Pheromones from a strange male mouse, particularly of a different strain, may prevent implantation or pseudopregnancy in recently bred females.
-The continued presence of the original male mate protects females from the Bruce effect (pheromone communication can occur in the absence of direct contact).
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Where does fertilization anatomically occur in mice?
In the ampulla or the upper portion of the oviduct. Ova can be fertilized to produce normal embryos for 10-12h after ovulation.
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Describe the reproductive changes during Proestrus.
Smear: epithelial to epithelial-cornified cells or epithelial-cornified cells; leukocytes to epithelial cells.
Uterus: hyperemia & distension increase; active mitoses in epithelium, few leukocytes.
Ovary & oviduct: follicles enlarged and distended with considerable liquor folliculi; few mitoses in germinal epithelium and in follicular cells.
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Describe the reproductive changes during Estrus.
Smear: Epithelial-cornified cells to cornified cells.
Uterus: distension and activity are maximal during estrus and then decrease. No leukocytes.
Ovary & oviduct: Ovulation occurs, followed by distension of the upper end of oviduct; active mitoses in germinal epithelium and in follicular cells.
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Describe the reproductive changes during Metestrus.
Smear: Cornified ++ cells, epithelial cells, leukocytes ++
Uterus: distension decreased; leukocytes in epithelium; walls collapsed; epithelium degenerates; mitoses rare.
Ovary & oviduct: Follicles undergoing atresia; growing corpora lutea; eggs in oviduct; few mitoses in germinal epithelium and in follicular cells.
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Describe the reproductive changes during Diestrus.
Smear: epithelial cells, leukocytes, more or less mucus.
Uterus: pale in appearance, walls collapsed; epithelium healthy but contains many leukocytes; some secretion by uterine glands.
Ovary & oviduct: Follicles begin rapid growth toward the end of period.
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What is the gestation length in mice?
19-21 days.
Postpartum estrus - lactation can delay gestation because of delayed implantation - may prolong gestation up to 12-13 days in certain inbred strains. Litter size usually decreases as female age, so females are usually retired around 6 months of age.
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How long does lactation last in mice?
Usually lactate for 3 weeks. Milk production increases up to 12 days postpartum and then declines until weaning at 21 days.
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Oxytocin is required for what aspects of mouse reproduction?
Oxytocin is required for nursing, but is NOT essential for parturition or reproductive behavior.
191
Describe the effects of the following male mouse pheromones:
1) Male-specific major urinary protein MUP20 (dacrin)
2) Male lacrimal protein ESP1
3) Exocrine-gland (lacrimal) secreting peptide 22 (ESP22) produced by juvenile male mice.
1) Rewarding and attractive to females; intermale aggression; area avoidance
2) Lordosis in female mice
3) Inhibitory on older adult male mating behavior
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Define deme
Group of wild mice; low exchange of individuals between different demes.
Each deme consists of kin-related members with a high degree of natural inbreeding, higher mutation rates compared to other mammals, and a wide range of developmental flexibility based on early life experience.
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What is the social structure of a mouse deme?
A dominant breeding male, and a hierarchy of females, subordinate males, and juveniles.
194
Define releaser pheromone.
Elicits an immediate behavioral response.
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Define primer pheromone.
Mediates a slowly developing and longer-lasting endocrine response.
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Define signaler pheromone.
Conveys individual or group identity, or mediates parent-offspring recognition and mate choice.
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What are major urinary proteins (Mups)?
- Small soluble proteins called lipocalins, which bind small organic chemicals with high affinity and function as pheromone transporters and stabilizers, but also act as protein pheromones themselves.
- They are synthesized in the liver and excreted in the urine as well as the nasal mucosa, lacrimal glands, and salivary glands.
- Chromosome 4 contains a cluster of 21 Mup genes, plus a number of pseudogenes.
- Along with small peptides, MHC class I peptides, volatile chemicals, and sex hormones, communicate dominance, kinship, diversity, and gender.
- Each inbred strain possesses a characteristic array of signals and to a certain extent unique signals exist among individuals of the same strain.
- Mice excrete pheromones in urine, as well as from planter, salivary, lacrimal, preputial, and mammary glands.
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What anatomical structures in mice detect pheromones?
Sensory neurons in the vomeronasal organ, the olfactory epithelium, septal organ of Masera within the olfactory epithelium, and the Gruenberg ganglion located at the anterior end of the nasal cavity.
Neuronal signals are transmitted to the ganglion layer of the olfactory bulb, and then to the brain.
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Which sex of mouse excretes more Mups?
Males excrete significantly more Mups in the urine than females.
200
Define kairomones.
Chemical signals between different species (for example, cat and rat Mups invoke fear in mice).
201
Which particular Mup is a known human allergen?
Mus m 1 (Ag1 or MA1); is encoded by the Mup 17 gene.
| Mice can excrete 1-5 mg/ml of Mups in urine and they are potent allergens in humans.
202
Define the Vandenbergh effect.
Acceleration of puberty in juvenile females in response to male urine.
203
Define the Lee-Boot effect.
Suppression of estrus cyclicity in group-housed females that are isolated from males.
204
What are some positive physiologic effects of providing environmental enrichment to mice?
Cognitive enhancement, delayed disease onset, enhanced cellular plasticity, and associated molecular processes in animal models of brain disorders.
205
What are the primary, secondary, and tertiary lymphoid organs in the mouse?
Primary - bone marrow, thymus
Secondary - lymph nodes, spleen, intestinal Peyer's patches
Tertiary - those that form in other solid organs in response to an insult or microbial exposure - GALT, BALT, etc.
206
Define pathogen-associated molecular patterns (PAMPs)
Recognized by the innate immune system to rapidly respond to antigen insult, via extracellular or intracellular pattern recognition receptors (PRRs).
Examples: LPS, bacterial flagellin, ss and dsRNA, non-methylated DNA.
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Receptors for PAMPs include ___________
TLR4 (recognizes LRS), TLR3/7 (ss and dsRNA), TLR9 (DNA); NOD-like receptors (NOD1/2), and RIG-like receptors (RIG-I, MGA-5)
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Which T and B cells are considered innate-like cells?
B-1 lineage B cells and gamma-delta T cells, as they express rearranged antigen receptor but seem to respond in an innate-like manner
209
How are leukocytes identified and classified?
By sets of monoclonal antibodies (mAb) against uniquely expressed surface receptors, typically measured by flow cytometry
210
What is a cluster of differentiation (CD)?
Identification of unique receptors on leukocytes by one or more mAb of the same specificity leads to the assignment of a receptor name, as a 'cluster of differentiation' (CD).
Ex: T cells are differentiated into CD4 or CD8 cells.
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What is the function of CD4+ T cells (T helper cells)?
Recognize peptides presented in MHC class II and promote B-lymphocyte activation and activate and regulate cellular immune responses via secretion of differing cytokines
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What is the function of CD8+ T cells?
Recognize antigenic peptides presented in MHC class I and serve as cytotoxic cells during cell-mediated immune response where they can destroy infected cells.
213
Describe 5 classes of immunoglobulin (Ig) isotypes.
IgG: most abundant Ab class in the serum; divided into subtypes IgG1, IgG2a/c, IgG2b, and IgG3; polymorphisms exist on the Ig locus such that some mice produce IgG2a (BALB/c) and some product IgG2c (C57BL/6)
-IgG2a/c is dominant in intracellular bacterial infections.
-IgG1 is dominant in extracellular bacterial infections.
IgM: secreted short term after initial antigen exposure followed by other isotypes
-IgE: associated with parasitic infections and allergies
-IgA: highest produced Ig in mice; production occurs in lamina propria of mucosal tissues, from where IgA is actively transported in dimeric form onto the luminal surface of mucosal tissues as 'secretory' IgA
-IgD: only expressed on surface of B cells in mice.
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What do different allotypes of immunoglobulins represent?
ALLotypes represent ALLeles (polymorphism = IgA2a)
Ex: BALB/c and 129SV mice express the Igh-b allotype, whereas C57BL/6 mice express the Igh-b allotype. Recombinant inbred strains of mice exist for both BALB/c and C57BL/6, which harbor the reciprocal Igh locus - these mice are useful tools for tracking B cells following adaptive cell transfer via allotype-specific mAb.
215
Define chemokines.
Family of small (8-10kD), secreted proteins that act primarily as chemoattractants for specific types of leukocytes; regulate cell trafficking to sites of antigen encounter
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How are chemokines grouped?
According to the number of cysteines and disulfide bonds in the molecule
217
What is the term for T cells that interact with B cells?
T follicular helper cells (T-subscript-FH).
| Their cytokine profile directs B cells to secrete a particular Ig isotype
218
What does IFN-gamma cause B cells to secrete?
IgG2a/c
219
What does IL-4 lead B cells to secrete?
IgG1
220
How long does reconstitution of the hematopoietic system take in gamma-irradiated 'bone marrow irradiation' chimera mice?
~6 weeks.
| During this time mice are provided with antibiotic-containing drinking water to avoid infections.
221
What techniques are commonly used for serology viral surveillance in rodents?
ELISA, multiplex fluorescent immunoassay (MFI) for simultaneous detection of Ab to multiple agents, indirect fluorescent antibody (IFA) assay, or hemagglutination inhibition (HAI).
IFA and HAI are generally used for confirmation.
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What is MAP testing?
Mouse antibody production testing. Used historically to test biological materials for contamination by infectious agents. PCR is now commonly used.
