Mouse models of human disease: lessons learned and promises to come. ILAR 43 (2): 055.
[Reviewer's Note - Any parts of this introductory article which recapped specific articles published in this ILAR issue were not reviewed as other LABSG members will be summarizing these articles for the LABSG list.] Many features of the mouse make it an ideal mammalian model for various human diseases. Some of these features include 1) novel technologies to allow manipulation of the mouse genome, 2) wealth of resources, inbred strains, and mutant stocks available to perform genetic studies in mice, and 3) the mouse's relatively short generation time and reasonable housing costs. However, the mouse is not the perfect model for everything. For early development and organogenesis studies, lower vertebrates (zebrafish, Xenopus) have the advantage in that early embryogenesis and differentiation occur outside the mother and can be easily viewed. Genetic mapping and genome manipulation are now being developed in these valuable lower vertebrate models. For some complex human disorders, we must choose between the "humanized" mouse and another model system. One of the lessons learned from mouse models is the difference in or lack of phenotype for the same enzyme deficiency in the mouse compared with human. Although a lack of phenotype may prove initially frustrating, it could be useful in exploring the consequences of alternate biochemical and physiological pathways and ultimately in developing therapies for such metabolic disorders. Mouse models are proving useful in understanding the pathogenesis of Mendelian single gene disorders as well as more complex disorders. Some common human diseases (mental illness, autism, Alzheimer's) do not occur naturally in the mouse, yet through genetic technologies, we are able to recapitulate disease features in mouse models. An advantage of mouse models is the ability to dissect out parts of a complex human disorder and study the contributions of individual components to the complete phenotype. The influence of environmental factors on a genetic trait can also be studied under controlled conditions in mouse models. For a geneticist, the raw starting material is variation. Initial discoveries in mouse genetics were related to variations among different strains or spontaneous or induced mutations. Later, additional mutant phenotypes were generated via large X-irradiation programs and chemical mutagenesis, and more recently, through transgenesis and homologous recombination in embryonic stem cells.
1. How many chromosomes does the mouse have?
2. Histocompatibility loci control expression of cell surface molecules that modulate major immunological phenomena, such as recognizing foreign tissue. What is the name of the major group and it's gene location?
a. H-1, chromosome 11
b. H-1, chromosome 13
c. H-1, chromosome 17
d. H-2, chromosome 13
e. H-2, chromosome 17
3. What is the average length of gestation in the mouse?
a. 15-18 days
b. 24-26 days
c. 21-22 days
d. 19-21 days
4. Which hormone promotes gametogenesis in both sexes of the mouse?
a. Luteinizing hormone
b. Follicle stimulating hormone
5. Which hormone promotes the secretion of estrogen in the female and androgens in the male?
a. Luteinizing hormone
b. Follicle stimulating hormone
6. Which of the following is descriptive of the mouse estrous cycle?
a. Polyestrous, 4-5 day length
b. Polyestrous, 10-12 day length
c. Polyestrous, 10-12 hour length
d. Polyestrous, 4-5 hour length
7. What is the number and anatomical location of the mouse mammary glands?
a. 6 pairs; three cervicothoracic and 3 inguinoabdominal
b. 5 pairs, two cervicothoracic and 3 inguinoabdominal
c. 5 pairs; three cervicothoracic and 2 inguinoabdominal
d. None of the above
8. What is the name of the effect when estrus is suppressed in mice housed in large groups due to pseudopregnancy or diestrus?
a. Bruce effect
b. Lee-Boot effect
c. Whitten effect
9. What is the name of the effect when a strange male mouse may prevent implantation or pseudopregnancy in recently bred females?
a. Bruce effect
b. Lee-Boot effect
c. Whitten effect
10. What is the name of the effect in which exposure to male pheromones restarts the estrous cycle and leads to estrus in most females' 3 days after pairing?
a. Bruce effect
b. Lee-Boot effect
c. Whitten effect
1) b; 2) e; 3) d; 4) b; 5) a; 6) a; 7) c; 8) b; 9) a; 10) c
Mouse models for disorders of mitochondrial fatty acid beta-oxidation. ILAR 43 (2): 057.
Mitochondrial beta-oxidation of fatty acids is required for energy production during periods of fasting and other metabolic stress. Human patients have been identified with inherited disorders of mitochondrial ß-oxidation of fatty acids with enzyme deficiencies at many of the steps in this pathway. Reye-like illness (hyperketotic-hypoglycemia, hyperammonemia, and fatty liver) and cardiomyopathy are common findings. Several mouse models have been developed to study these disorders. Models have been generated using both a "phenotype driven approach" (i. spontaneous mutations discovered by phenotype screening and, ii. radiation/chemical mutagenesis) and a "genotype driven approach" (gene targeted mutations).
The article contains a detailed figure illustrating the steps of mitochondrial fatty acid oxidation. An understanding of these steps in essential for comprehending the significance of these animal models but is beyond the scope of this summary. Briefly, the fatty acid is activated to the acyl-CoA form and transported into the mitochondria as an acyl-carnitine via a process requiring carnitine palmitoyltransferase-1 (CPT-1), canitine/acyl-carnatine translocase (CT), and carnitine palmitoytransferase-2 (CPT-2). There are two isoforms of CPT-1 designated CPT-1a and CPT-1b. Carnitine-dependent transport of long-chain fatty acids requires additional steps.
In a complete cycle of beta-oxidation, fatty acids are catabloized to generate and acetyl-CoA and acyl-CoA, which is two carbon units shorter. The first step within the mitochondrial matrix is the acyl-CoA dehydrogenation step, which is catalyzed by a group of enzymes named for the chain length of the substrate on which they act: very long-chain acyl-CoA dehydrogenase (VLCAD), long-chain acyl-CoA dehydrogenase (LCAD), medium-chain acyl-CoA dehydrogenase (MCAD), and short-chain acyl-CoA dehydrogenase (SCAD). Next, enoyl-CoA hydratase catalyzes the hydration of the double bond, which results in the formation of 3-hydroxyacyl-CoA. This process is followed by the 3-hydroxyacyl-CoA dehydrogenases (long-chain hydroxyacyl-CoA dehydrogenase [LCHAD] or medium-/short-chain hydroxyacyl-CoA dehydrogenase [M/SCAHD]), which catalyze the formation of 3-ketoacyl-CoA through an NAD+/NADH-dependent dehydrogenation of 3-hydroxyacyl-CoA. Finally, 3-ketoacyl-CoA thiolase catalyzes the reformation of the shorter acyl-CoA and the newly generated acetyl-CoA through a thiolysis reaction.
These last three steps for long-chain substrates are accomplished by long-chain mitochondrial trifunctional protein (MTP), which is composed of MTP alpha and beta proteins. MTP alpha and MTP beta are encoded by two separate but linked genes, HADHA and HADHB, respectively. MTP alpha contains the long-chain enoyl-CoA hydratase and LCHAD activities, whereas, MTP beta contains the 3-ketoacyl-CoA thiolase activity.
The products of beta-oxidation are used to generate the energy necessary for energy homeostasis. FADH2 and NADH are reoxidized by oxidative phosphorylation within the mitochondrion for ATP synthesis or heat production. Acetyl-CoA, which is generated, is either passed into the tricarboxylic acid cycle for the production of additional ATP or is used for the production of ketones. Ketogenesis is particularly important as an alternative fuel to the brain and heart during periods of starvation. All inborn errors of mitochondrial fatty acid beta-oxidation are characterized by fasting induced episodes of Reye-like illness.
The following is a description of defects identified in human fatty acid beta-oxidation:
1. Human mitochondrial carnitine/acyl carnitine transport defects. Although there have been patients described with CPT1a deficiency, there are no known patients with CPT1b deficiency. Overall, patients with CPT 1a deficiency have shown a prominent hepatomegaly with fatty change; however, there was no cardiac disease, and plasma carnitine concentrations often were not elevated. CPT-1a-deficient patients are treated with dietary management strategies such as frequent feedings with medium-chain triglycerides and strict avoidance of fasting.
2. Human VLCAD deficiency. There have been many human patients identified with VLCAD deficiency. These patients commonly present during infancy or childhood with cardiomegaly or skeletal myopathy in addition to the common Reye-like illness. Instances of sudden death in infancy also have been attributed to VLCAD deficiency. Mutations of the VLCAD locus (ACADVL) are heterogeneous. Although LCAD deficiency was originally described in human patients, it turned out to be a mistaken diagnosis and most of those patients were eventually shown to be VLCAD deficient. Thus, there have been no documented cases of human patients with LCAD deficiency.
3. Human MCAD deficiency. MCAD deficiency is the most common of the human acyl-CoA dehydrogenase deficiencies. Disease episodes are characterized by acute metabolic acidosis, hypoketotic hypoglycemia, hyperammonemia, secondary carnitine deficiency, and medium-chain dicarboxylic aciduria. In contrast to VLCAD deficiency, cardiac disease is not a common feature in MCAD-deficient patients.
4. Human SCAD deficiency. SCAD-deficient patients have a variable phenotype ranging from neonatal death to a single life-threatening episode followed by complete recovery. During acute metabolic crisis, SCAD-deficient patients experience metabolic acidosis, hypoketotic-hypoglycemia, and short-chain dicarboxylic aciduria characterized by increased excretion of ethylmalonic and methylsuccinic acids.
5. Human Mitochondrial Trifunctional Protein (MTP) and Isolated LCHAD Deficiency. There have been reports of human patients who have MTP deficiency, which includes LCHAD deficiency. In contrast, there have been other patients with isolated LCHAD deficiency. LCHAD deficiency, like the other diseases of this pathway, often occurs in infants characterized with Reye-like illness. These patients also may have hypotonia and cardiomyopathy. In addition, there are several LCHAD-deficient patients described with retinopathy, cirrhosis, and peripheral neuropathy, characteristics not found in other disorders of ß-oxidation. LCHAD deficiency has been implicated in acute fatty liver of pregnancy (AFLP) and the hemolysis, elevated liver enzymes, low platelets (HELLP) syndrome. AFLP has most often presented with jaundice, abdominal pain, vomiting and lethargy. Patients with AFLP have had signs consistent with acute liver and renal failure that resolved after delivery of the fetus. Patients with HELLP syndrome also experienced abdominal pain as well as headache and hematuria.
6. Synergistic heterozygosity. In these cases, there would be heterozygous deficiencies in two or more different enzymes within a similar pathway such as fatty acid ß-oxidation. Specifically, these patients would suffer from haploinsufficiency of two different enzymes resulting in a net functional deficiency in that metabolic pathway, with a disease severity like that of a homozygous deficiency at a single enzymatic step.
Mouse Models of Disorders of Fatty Acid Beta-Oxidation:
1. Mitochondrial carnitine/acyl-carnitine transport defects. The genes for mouse CPT-1a, CPT-1b, and CPT-2 have been cloned and mapped. A mouse model is currently under development for CPT-1a deficiency. At the time of this writing, no other reported mouse models have been developed to investigate inherited deficiencies of CPT-1b, CT, or CPT-2.
2. VLCAD deficiency. Two mouse models of VLCAD deficiency have been produced independently. In the model described by Cox, et al, clinical disease includes mild hepatic steatosis, mild fatty change in the heart in response to fasting, and cold intolerance.
3. LCAD deficiency. Although there are no known human LCAD-deficient patients, an LCAD-deficient mouse has been developed that exhibits the most severe disease phenotype compared with the mouse models for any of the other acyl-CoA dehydrogenase deficiencies. These mice are characterized by sudden death, gestational loss, fatty change of the liver and heart, and cold intolerance. LCAD-/- mice have been crossed with leptin-deficient obese mice. It was expected that the combination of the obese phenotype with LCAD deficiency would exacerbate both phenotypes; however, there were no major differences in severity of phenotype in the double mutants compared with the individual phenotypes.
4. MCAD deficiency. There is a mouse model for MCAD deficiency in development.
5. SCAD deficiency. SCAD deficiency was discovered in BALB/cByJ mice. Young SCAD mutants develop a fatty liver coupled with hypoglycemia when fasted overnight. Homozygotes are cold intolerant. The SCAD-deficient mouse model was used in studies pursuing gene therapy correction of beta-oxidation defects. Phenotypically, the treated mice had a decreased incidence and severity of fatty liver and a decreased organic aciduria metabolite pattern, even after a fast.
6. Mitochondrial trifunctional protein and LCHAD deficiency. Mitochondrial trifunctional protein (MTP) constitutes three enzyme activities of fatty acid beta-oxidation. A knockout mouse model has been developed for mitochondrial trifunctional protein deficiency by gene knockout of MTP alpha gene (Hadha). This condition in the mouse is lethal during the neonatal period, and the pathogenesis includes hepatic steatosis, myocyte necrosis, and hypoglycemia.
7. M/SCHAD deficiency. M/SCHAD knockouts were generated using gene-targeting techniques. Like many other mouse models of mitochondrial fatty acid ß-oxidation, these mice are cold and fasting intolerant. They also develop hepatic steatosis.
Practical Considerations. When maintaining colonies of mice with disorders of FAO, care must be taken to ensure reduced metabolic stress, including variations in ambient temperature (particularly the avoidance of drops in temperature) and periods of inadequate amounts of food. In addition, when considering the shipment of these mice, it is important to take into consideration the time of year (avoid cold temperatures) and the availability of food and water sources. These mouse models are extremely susceptible to any perturbation in homeostasis
1. Reye-like illness is characterized by fasting induced episodes of all of the following EXCEPT:
b. Metabolic Alkalosis
d. Fatty liver
e. All of the Above
2. True of false: The LCAD deficient mouse has a phenotype that closely approximates human LCAD deficiency.
3. Which of the following is an important consideration in the husbandry of mice with disorders of fatty acid oxidation?
a. Variations in ambient temperature
b. Periods of inadequate amounts of food
c. Time of year and temperature for shipping animals
d. All of the above
2. False, human LCAD deficiency has not been reported.
The mousetrap: what we can learn when the mouse model does not mimic the human disease. ILAR 43 (2): 066.
This review article outlines examples of human diseases which cannot be mimicked in a mouse model with a simple gene knockout or double knockout. In the article there are examples of diseases that reproduce some of the human disease phenotype, may be more severely affected than the human disease, or have no clinical phenotype at all. The article discusses four x-linked disorders and four autosomal disorders.
X- Linked disorders:
Lesch-Nyhan Syndrome: deficiency of the hypoxanthine-guanine phosophoribosyltransferase ( HPRT) in the purine salvage pathway.
In humans this deficiency of HPRT results in overproduction of uric acid, debilitating neurological changes, cognitive disability and impulsive and self-injurious behaviors. Decreased dopamine levels in the basal ganglia and CSF have also been reported in these patients. two mice models of Hprt deficiency have been created. In both models male mice have a total lack of HPRT , however they act normally and have no apparent phenotype. The mice also have a decrease in dopamine in the basal ganglia but not to the extent humans do.
It appears that the mouse must have an unidentified alternate route for purine synthesis. In the knockout mice purine content in the brain is normal, de novo purine synthesis is accelerated.
Lowe Syndrome: This disease in humans is manifested by mental retardation, renal Fanconi syndrome, and congenital cataracts. The gene responsible for Lowe syndrome is OCRL1, which encodes for phosphotidylinositol-4,5-biphosphate 5 phosphatase, which is necessary for inositol metabolism. A mouse model knocking out the Ocr11 gene resulted in mice with no Ocr11 enzyme activity but no apparent phenotype. Mice were studied using a variety of biochemical and behavioral parameters. A mouse knockout of Inpp5b was created to see if this enzyme ws compensating for the Ocr gene deficiency. No phenotypic abnormality was expressed. At sexual maturity the male mice had testicular degeneration. A double knock-out missing the OCr1 and the INPP5b resulted in 100% embryonic mortality.
X-Linked Adrenoleukodystrophy: In the human disease affected males accumulate unusually high levels of unbranched very long-chain fatty acids ( VLCFA's) in their brain and adrenal cortex. The gene for X-ALD is a member of the ATP-binding cassette transporter family The mouse X-ALD gene maps to Band B of the mouse X chromosome. Three groups have created a mouse that has no X-ald gene and they all have very high levels of VLCFAs, however no clinical or neurological phenotype has been observed. The biochemical study of these mice indicates that there must be some differences between mouse and human in metabolism or processing VLCFAs. An understanding of these differences could also lead to a discovery of a treatment for the human disease.
Fabry Disease- Alpha-Galactosidase deficiency:
This disease in human males results in an accumulation of glycosphingolipids in the lysosomes of perithelial,
endothelial, ands smooth muscles cells of blood vessels as well as the ganglion and other tissues of heart and kidney. hemizygous males present with angiokeratoma, painful paresthesias,corneal dystrophy, renal impairment, gi pain and diarrhea, as well as cardiac disease. In 1997 a mouse model of Fabry disease was made by disruption of the mouse gene Gla by homologous recombination in ES cells. All of the mouse models lack the a-gal A enzyme and have extensive lipid accumulation in the liver and kidney. The mice are like the humans biochemically and pathophysiologically, but they exhibit none of the clinical phenotypes that the humans lacking this gene exhibit.
Galactosemia: This disease results from a enzyme deficiency of any of three enzymes involved in the metabolism of galactose.Classic galactosemia results from the deficiency in galactose 1- phosphate uridyltransferase( GALT). This presents early in life as failure to thrive, vomiting, diarrhea, jaundice. Congenital cataracts, liver dysfunction, renal tubular acidosis and septicemia are also often identified. Mice deficient in GALT were produced by removal of 6-8 exons of the GALT gene. Biochemically these mice are similar to humans, but they do not
show any of the toxic effects humans exhibit with a GALT deficiency. One theory is that galactose transport across the blood-brain barrier may differ from mice and humans. Mice could only be created to develop galactosemic cataracts
when crossed to mice that overexpressed aldose reductase.
GLYCOGEN STORAGE DISEASE TYPE II, Pompe disease:
this is an autosomal recessive disease which leads to abnormal lysomal accumulation of glycogen in the tissues of affected individuals. In humans the disease is severe in infant onset type, characterized by hypotonia, cardiomegaly, hepatomegaly and fatal cardiorespiratory failure by age 2. the adult onset disease is characterized by progressive myopathy in the skeletal muscle and diaphragm.
Two separate groups developed a mouse model, one by disrupting the murine alpha-glucosidase gene through insertion of a gene in exon 6, the other model was created by deleting exon 6. the two models are biochemically similar, the phenotypes quite different. The mice with the gene inserted at exon 6 accumulated glycogen in the skeletal muscle and developed progressive weakness and early as 3.5 weeks of age and muscle wasting. The mice with the exon 6 gene deletion also accumulated glycogen in skeletal muscles and heart but did not exhibit locomoter abnormalities until 6 months of age. The genetic background was then evaluated to see what role it played in the disease. The exon 6 was deleted on mice with a background of 129 x C57BL/6 and it resulted in muscle wasting and cardiomyopathy. When the exon 6 was deleted on mice with a 129 x C57BL6 x FVB background the mice developed a milder phenotype and later onset of disease. these models do provide a means to test potential therapies for GSDII.
Initial studies using human recombinant protein in knockout mice resulted in significant correction of the enzyme deficiency in all tissues except brain and a decrease in lysosomal glycogen storage.
Metachromatic Leukodystrophy- This is another autosomal recessive lysomal storage disease in which the glycolipid cerebroside-3-sulfate accumulates in the cells of affected tissues due to a deficiency in the enzyme arylsulfatase A. the clinical
manifestations are variable, but most patients display widespread demyelination and progressive degenerative neurological symptoms. The human arylsulfatase A gene( ARSA) maps to 22 and the mouse ortholog( As2) maps to mouse chromosome 15.
In 1996 Hess created an As2( -/-) mouse using gene targeting. These mice however were phenotypically mild compared to human patients. Mice exhibited neuronal damage in the ear causing deafness, but widespread demyelination did not occur as does in humans.
Tay-Sachs and Sandhoff Diseases( Gm2 Gangliosidoses)
These two diseases are autosomal recessive lysosomal storage disorders that involve the stepwise hydrolysis of the ganglioside
Gm2 through the action of beta hexosaminidase enzyme ( Hex) of which there are two isoenzymes Hex A and Hex B.
Tay- Sachs results from mutations in Hex A and Sandhoff's Disease results from mutations in Hex B. Both of these defects result in accumulation of Gm2 storage material in neurons causing profound neurological degeneration.
Hexa and Hexb knockout mice were created.T the Hexa(-/-) were created with gene targeting by Yamanaka in 1994. These mice retained less than 1 % of normal Hexa activity. the brain of these mice progressively accumulated the Gm2 ganglioside, however they did not exhibit any clinical phenotype and were found to be normal in balance, behavioral and motor patterns. In 1995 a Sango et al created both hexa and hexb knockouts.They found their Hexa to be just like the Hexa knockouts made by Yamanaka. The hex b(-/-) mice displayed progressive deterioration in motor function and gait abnormalities by 12 to 16 weeks.
they further pursued the gangliosides present in the Hexa and Hexb mice. In the Hexa mice only Gm2 was present. In the Hexb mice Gm2 and Ga2 were both greatly elevated in the knockout mice.Further analysis of the pathway in mice led the researchers to conclude that there are two independent pathways for the degradation of Gm2. The TS and Sandoff murine models have been very useful in developing more information about the pathway for ganglioside degradation in mice and humans.
Other diseases mentioned at the end of the article, cystic fibrosis( CF) many murine models have been made but none of them completely mimic the human disease. Another disease, Gaucher disease, demonstrates the most severe form of the disease in the mouse model. The take home message: simply recreating a metabolic disruption in a mouse does not always give you the same clinical phenotype seen in the human.
1.Which disease is not an x-linked disease?
c. Pompe disease
d Fabry disease
2. In which of the following diseases is the mouse model more severe than the human disease?
a. Gaucher disease
b. Lowe syndrome
c. Tay- Sachs disease
d. Lesch-Nyan disease
3. In which of the following disease models do the mice become deaf?
b. Metachromatic Leukodystrophy
c. Glycogen Storage Disease II
1. C. Pompe disease 2. A. Gaucher disease 3. B. Metachromatic Leukodystrophy
Complexities of cancer research: mouse genetic models. ILAR 43 (2): 080.
This article focused primarily on quantitative trait genetic analysis and tools that may be used in learning more about cancer genetics. It was noted that we currently have a very good understanding of the high-risk, familial cancers. However, the numbers of other cancers that are not in this group represent the majority of cancers, and their causes are presumably multifactorial. The focus of cancer is this review is minimal. Some key research discoveries that have contributed to our understanding of cancer genetics are the following:
· Oncogenes- genes whose products promote cell proliferation
· Tumor suppressors- function to inhibit or check cell proliferation; inheritance of an inactivated allele of a suppressor gene (the first hit) increases the risk of cancer. The stochastic somatic inactivation of the second allele (the second hit) then triggers uncontrolled proliferation (examples of this model include the retinoblastoma gene and protein, WT!, NF1 and p53)
· Genetics of cancer research- cancer is a family of related diseases, not a single disease. The characteristic of unregulated cellular proliferation is common among all cancers, but the genes and the genetic and genomic events leasing to neoplasia are different for each tumor type.
With the human genome being sequenced the inherited cancer syndromes should soon be identified. Then, the challenge for cancer genetics is to move forward from the Mendelian genetics of the rare familial cancer syndromes into the field of quantitative trait loci (QTLs), susceptibility factors and modifier genes. This type of analysis is not unique to cancer, and is common to all complex trait genetic analysis. Critical to this type of evaluation is correct phenotyping. Human studies are not practical. Therefore, in the foreseeable future, the rodent models are the workhorses for QTLs. The advantages of using rodents were: their small size, low relative cost, short gestation, inbred strains and genetic diversity available, and the ability to manipulate their genome at both the individual nucleotide and the gross chromosomal level. The utility of the large mutagenesis programs in cancer research are limited due to the later onset of cancer in these models, making systematic evaluations burdensome. Ethylnitrosourea mutagenesis is still important for cancer research, as well as behavioral and developmental pathways. However, early onset tumors will likely be due to familial inheritance, and will not be helpful with the identification of tumor susceptibilities that are present in the bulk of the human population or with the weakly penetrant cancer phenotypes. The environmental component of cancer susceptibility is still lacking.
The vast majority of cancer in the human population is due to the intersection of genetic predispositions caused by polymorphisms in metabolic or regulatory genes and exposure to carcinogenic environmental agents. Genetics and environment both can influence tumor incidence. The experimental crosses of strains of mice known to differ significantly in tumor susceptibility and gene-environment interactions are practical tools for cancer research. A variety of different methods have been developed or adapted to extract gene loci that modify cancer development. The most commonly used strategy is mapping in backcross or intercross mapping. Strategies also include combinations of recombinant inbred mapping panels and experimental crosses, recombinant congenics, chromosomal substitution strains, recombinant inbred segregation tests, and recombinant inbred intercrosses. These methods were discussed and their weaknesses and strengths were outlined.
Backcross and Intercross Analysis
· This is the most common form of QTL
· F2 intercross- generates an idea of the approximate number of modifier loci present and obtains estimates of their additive or dominance effects; would require approximately 30% fewer animals than a backcross.
· Backcross- efficient in situations in which few loci are known to have dominance effects.
· STRENGTHS: any two genetically distinct strains of mice can be used to generate a mapping cross. Thus, the genetic components of any phenotype that differ between two of the multitude of inbred mouse strains can be subjected to genetic dissection.
· DISADVANTAGES: lack of resolution (measuring in centimorgans rather than sub centimorgans as required for positional cloning). Preliminary genome localization of QTLs by this method must be supplemented with fine mapping analysis which takes a tremendous amount of time and animals to achieve the desired goal.
*RIs, RCs, Congenics and CSS have defined and fixed genotypes.
Recombinant Inbred Mapping
· Recombinant inbred (RI) strains have been used to map a wide range of Mendelian loci and quantitative traits which is particularly useful when there is low heritability.
· STRENGTHS: each recombinant genome is replicated in the form of an entire isogenic line. The variance associated with uncontrolled error can be suppressed to very low levels which elevate heritability and improve prospects for mapping QTLs. Can be used to exploit gene: environment interactions.
· DISADVANTAGES: RI sets exist for relatively few key inbred strains. As a result, on the subset of polymorphic loci that differ among these few groups of inbred mouse strains can be mapped using this strategy. (RI sets exists for C57Bl/6J, A/J, Balb/cJ and DBA2/J)
Recombinant Congenic Analysis
· Recombinant congenics (RC) panels are composites of the genomes of two inbred progenitor parent strains, just like the RI panel. Unlike the RI, which are generated by serial brother-sister matings after an initial outcross, two rounds of backcrossing to one of the progenitor strains precedes the brother-sister mating to achieve genome homozygosity.
· STRENGTHS: The resulting RC strains contain a reduced amount of the donor strain genome (12.5%) compared with backcross, intercross or RI analysis. The reduction in the amount of donor genome present in the RC compared with the RI lines increases the probability of separating interacting genes into individual strains where their effects can be studied in isolation.
· DISADVANTAGES: Unlike the RIs, RCs are time consuming to make and only traits that differ between the progenitor strains can be assayed.
· One of the most common strategies to validate QTLs where high and low alleles are swapped between the low and high parental strains. If the QTL is real and has been mapped to the correct interval, then phenotypes of the reciprocal congenic lines should each deviate from the recipient strains by a predictable amount.
· STRENGTHS: usually converts a polygenic trait to a monogenic QT.
· DISADVANTAGES: Each QTL needs its own mouse resources- tremendous amount of genotyping and 5 (speed congenics) to 10 (standard congenics) generations of backcrossing.
Chromosomal substitution strains (CSS)
· A variation of the congenic strain where a whole chromosome from the donor strain is in the background of the recipient strain.
· STRENGTHS: The CSS increases the power to detect QTLs because the variance due to environmental or experimental fluctuations can be averaged out by repeated analysis of a genotype.
· DISADVANTAGES: The QTL assignment is to a whole chromosome, rather than a chromosomal region. Cannot distinguish between single and multiple QTL spm the donor chromosome.
· An extension of the RI which relies on a potentially large set of diverse F1 intercrosses (X) generated from many different pairs of RI strains.
· STRENGTHS: shares all the benefits of RI, with the added benefit of providing a very large sample of unique and predictable genotypes. There is hybrid vigor of the F1 (as with all F1s) which is used to determine phenotypes and the ability to make each F1 by reciprocal crosses, which is a feature that can partly control for parental effects.
· An RI can be backcrossed to one of many more inbred strains to generate recombinant backcross (RIB) progeny. Unlike RI, the RIB crosses share segregation ratios similar to those on standard backcrosses, with a 1:1 ratio of homozygous to heterozygous genotypes. The het genotype component of this model is not available in the RI.
1. What do the following acronyms stand for:
2. T/F Most human cancers are of familial origin.
3. What species of laboratory animal is being used primarily in QTL analysis.
1. QTL-quantitative trait locus
SNP-single nucleotide polymorphism
2. False- most are polygeneic and affected by environmental influences.
3. B. Mouse
Mouse models of Alzheimer's disease: a quest for plaques and tangles. ILAR 43 (2): 089.
The majority of Alzheimer's Disease (AD) cases are sporadic but, 10% of the cases are familial. The familial cases have permitted important insights in the disease such as mutations in the genes encoding amyloid-B precursor protein (APP), presenilin (PS1), and presinilin 2 (PS2), which are associated with early onset autosomal dominant AD.
Morphologically, the brain is atrophic in AD - most pronounced in the frontal, temporal, and parietal lobes. There is also a symmetrical dilation in the ventricular system which reflects a generalized loss of brain parenchyma. Microscopically, AD is characterized by the presence of filamentous protein aggregates (neurofibrillary tangles) within the cytoplasm of neurons in the neocortex, hippocampus, basal forebrain, and some areas of the brainstem. Neurofibrillary tangles are made of neuritic processes that contain insoluble helical filaments These filaments are primarily composed of hyperphosphorylated tau protein. Tau is one of the microtubule-associated proteins that stabilizes neuronal microtubules. Plaques form when accumulations of the tau-enriched helical filaments occur within distal neuronal cell processes (neurites) and are found in the neuropil of the cerebral cortex and hippocampus. The senile plaques also contain a central core of amyloid B protein (AB). The presence of neurofibrillary tangles and/or senile plaques is not entirely specific for AD - identical structures are frequently present in brains of cognitively normal elderly people. Rather, it is the density and widespread distribution of these changes that leads to a diagnosis of AD.
Mouse models have been developed by manipulation of APP, PS1, PS2, and tau since it is these molecular components that are thought to be associated with the incidence of AD.
There are 5 categories of transgenic mouse models of AD. The following list includes examples of each:
I. Amyloid-B Precursor Protein (APP):
a) PDAPP Mouse: this mouse expresses a human APP mini gene on a mixed C57BL/6, DBA, and Swiss-Webster strain background. The success of this model is associated with the high level of APP expression achieved; extensively used model. Although this model developed amyloid deposits, it failed to meet all criteria of human AD due to the lack of cortical/hippocampal neuronal loss and absence of neurofibrillary tangles.
b) Tg2576 Mouse: expresses human APP at a level more than 6-fold higher than endogenous murine APP; on C57B6/SJL background. Amyloid deposits at 6-9 mos of age though no neuronal loss and no neurofibrillary tangles.
c) APP23 Mouse: Amyloid plaques develop at 6 mos of age. There is substantial neurodegeneration and hyperphosphorylated tau though no neurofibrillary tangles developed.
Conclusions: The above models have the important feature of plaque formation that parallels human AD. However, none of these models reflects the complete picture of AD because neurofibrillary tangles are not identified and prominent neurodegeneration and cerebral atrophy do not occur.
II. Presenilin Transgenic Mice:
d) PS1 Null Model: Embryonic lethality; embryonic studies indicate that expression of amyloid B protein in brain is increased.
e) PDF Promoter Model: a second PS1 transgenic model offered similar results. Brain homogenates indicated increased levels of amyloid B protein.
III. Tau Transgenic Mice:
f) Human Tau, shortest isoform (T44): expresses high levels of human tau. These mice developed insoluble intraneuronal filamentous hyperphosphorylated tau inclusions by 12 mos of age. Also, developed features of another disease type (non-AD) such as axon degeneration, spinal cord gliosis and motor weakness.
g) JNPL3 Mouse: expresses human four-repeat tau. Tau-immunoreactive pre-tangles were found but at a lower level than commonly found in human AD. The tangles were associated with neuronal degeneration, especially in the spinal cord.
IV. Apolipoprotein E Transgenic Mice:
h) Tg2576xApoE Mouse Model: reduced amyloid B protein as compared to the Tg2576 mouse model; no neurodegeneration.
i) PDAPPxApoEMouse Model: character and distribution of amyloid deposits changed which implicates ApoE in maturation of amyloid plaques.
V. Neprilysin Transgenic Mice:
j) Neprilysin-null Mice: this model offers evidence that neprilysin (protease) could be a natural amyloid B-degrading enzyme. Relevance of neprilysin to AD in humans was suggested when it was discovered that there are low levels of neprilysin in plaque regions in patients who died of AD.
Conclusions: None of the mouse models to date recapitulate the complete the neuropathology of AD. However, the genetically altered mouse has offered tremendous insight into the various molecular elements in the pathogenesis of AD.
1) T/F: The presence of neurofibrillary tangles and/or senile plaques are associated with fulminant clinical AD.
2) Which of the following are features that may be associated with development of AD:
a) Mutations in gene encoding Amyloid - B Precursor Protein (APP)
b) Mutations in gene encoding Presinilin 1 (PS1)
c) Mutations in gene encoding Presinilin 2 (PS2)
d) Accumulation of hyperphosphorylated tau within degenerating neurons e) All of the above (A: choice "e")
1. (A: false - these structures are also frequently present in cognitively normal elderly people. It is the density and distribution of these changes in the cerebral neocortex that lead to AD)
Welfare issues of genetically modified animals. ILAR 43 (2): 100.
Genetically engineered animals have opened new frontiers in biomedical research but also present many challenges in reviewing protocols and providing adequate care.
Modification of the genome of animals has occurred throughout the ages. Initially, spontaneous genetic changes occurred. Later, humans selected animals with desirable genetic traits and purposeful utilized them for breeding. More recently, genetic engineering evolved. It comprises transgenic animals created by injection of foreign DNA into the pronucleus of embryos, knockout animals developed by ablation of a gene sequence of interest in embryonic stem cells, and point mutagenesis accomplished by administration of e.g. N-ethtyl-N-nitrosourea (ENU) or chlorambucil.
Areas of research that benefit from the creation of genetically engineered animals are:
- Gene discovery: determines the structure and functions of various genes, proteomics a related area studies proteins produced by genes, their actions, and interactions with other proteins in the body.
- Disease Models: creation of new improved models for human disease, e.g. research into the genetic influence on tumor promotion and suppression by overexpression or inactivation of specific genes.
- Test System Development: improved test systems for examining safety and toxicity of chemicals, products, drugs, and devices; includes carcinogenicity testing, drug safety and efficacy evaluation etc.
- Gene Therapy: treating disease by altering the genome of somatic cells of humans and animals, e.g. replacement of a defective gene with a normal one, insertion of resistance genes, or altering regulatory sequences to turn genes on or off.
- Xenotransplantation: demand for transplantable organs fosters research into creation of animals with tissues compatible for implanting into humans.
- Life Span Extension: advances in control of disease results in increase of longevity in humans; line of mice with a knocked out proto-oncogene at the SHC locus raises hope of prolonged life span even further since p66shc-/- mice show increased resistance to oxidative stress aAdvantages of genetic engineered animals:
- More accurate models with provision of more precise data than previously used paradigms, e.g. B6SJL-Tg N (SOD1-G93 A) mice overexpressing mutant CU, Zn superoxide dismutase to mimic amyotrophic lateral sclerosis (ALS).
- Permits replacement of higher species with less sentient species, e.g. transgenic mice expressing the human poliovirus receptor may substitute honhuman primates to test the safety of attenuated polio vaccines.
- Possibility of reduction of the number of animals required addressing a particular disease due to more precise models.
Welfare issues of genetic engineered animals:
- Genetically altered animals may present challenges to the IACUC in evaluation of newly created lines, their special requirements for maintaining health and well-being.
- Unforeseeable health/animal welfare issues:
- - Genes to be studied (overexpressed or knocked out) may be necessary for fetal development or basic functions of life, consequently produce health problems, premature lethality, and shortened life span of those animals.
- - Models of induced human diseases elicit same symptoms in animals as in humans, especially welfare issue in untreated control animals, e.g. B6SJL-Tg N (SOD1-G93 A) mice used for study of ALS develop hind limb paresis progressing to paralysis.
- - Mice engineered for oncology studies may suffer due to tumor burden, e.g. strains of mice knocked out for transforming-related protein 53 tumor suppressor gene with increased incidence of tumors and its negative consequences.
- - Manipulations of the immune system of animals, e.g. to render their tissue compatible for human xenotransplants, lead to decreased disease resistance and altered susceptibility to microorganisms, subsequently represent some novel diagnostic and therapeutic challenges.
- Welfare concerns in production and breeding:
- - Most controversial and vexing issue is the sharp increase in numbers of mice in recent years in opposition to the proposed reduction of animals due to genetic engineering.
- - This is largely attributable to intensified research in areas that were previously hampered by a lack of adequate models.
- - Considerable numbers of animals are necessary to create each genetically modified line, breeding males and donor females needed to produce embryos for pronuclear injections and for harvesting blastocysts for injection of modified embryonic stem cells, vasectomized males needed to breed with females to produce pseudopregnant recipients for the altered embryos.
- - Only <1% to 10% of the offspring of males treated with ENU for point mutagenesis have interesting phenotypes, up to three generations of breeding and screening are required to detect mutant progeny.
- - All animals used to produce the genetically modified line must be bred to generate animals to study. The breeding animals themselves do not deliver usable research data. Emerging nontransgenic and wild-type littermates are not suitable for research or further breeding, will be euthanantized.
- - Simply to maintain a line, mice with compromised health may be bred but not studied.
- Unpredictability of welfare problems in newly created lines:
- - Phenotypical outcomes in transgenic mouse lines will vary due to the randomness of the process of incorporating the DNA construct into the genome even when the same methodology and identical DNA constructs are utilized. Phenotypes are influenced by the number of copies of the same DNA construct inserted, by the incorporation of the same DNA construct into a different chromosome (even a different location on the same chromosome), by variations in regulatory sequences activated or inactivated, and differences in the background strain of mouse.
- - There is a large variation of phenotypes in mouse strains produced by ENU mutagenesis, too due to randomness or variation in which the particular genes are mutated.
- Virus vectors used as carriers of genetic material for gene therapy are accompanied by the concern of causing welfare problems in recipient animals even if replication-defective. If a helper virus were available, the vector virus might regain the ability to replicate and transmit the transgene to other animals or humans. The effect of an underlying disease on the host's susceptibility to the virus vector must also be considered.
- The welfare of feral populations and the environment must be respected. Escape of animals with stable introduction of recombinant DNA into their germ line and breeding of those with feral populations could alter the environment and create a disastrous situation.
Tools to identify, prevent, or limit animal welfare concerns:
- Search of databases by an investigator or an IACUC reviewer to investigate what is already known regarding lines of genetically engineered animals that have been characterized previously. The transgenic animal/targeted mutation database TBASE (<http://tbase.jax.org>) provides information about lines of transgenic and targeted mutant mice, reports the age of onset of changes in phenotype produced by the mutation, progression of disease conditions, and points at which euthanasia should be considered. Other relevant databases are available through the Mouse Genome Informatics web site (<http://www.informatics.jax.org>).
- A clinical surveillance system is especially necessary if new and uncharacterized lines will be utilized. The frequent observation of animals to identify concerns early is as important as a reporting system for veterinary evaluation of morbidity and mortality. The detection and assessment of illness, physical deficits, injury, or abnormal behavior should be shared duties of animal care, research, and veterinary staffs alike. Even seemingly insignificant changes should be studied and documented as potential effects of the genetic alteration. This can also be helpful in designing monitoring protocols for established lines since not always all phenotypical signs are listed at the available databases. The surveillance system should also include regular evaluation for murine pathogens, serology, necropsy, and histology.
- The phenotype assessment is the responsibility of the research team. When gross lesions appear, a careful necropsy, including histology should be a component of any basic phenotyping protocol. A more specialized testing is the behavior phenotyping.
The results of the phenotype assessment should be available to the IACUC review when an investigator requests approval for continued breeding of a line.
- Institutions conducting biomedical research involving genetically modified animals are
obligated to take containment measures to prevent the escape of those animals and establish programs to prevent feral rodents from gaining access to the animal facilities through establishment of BL1-N conditions (Federal Register 1994). Live mice that are genetically altered should not be released to zoos or pet stores to be used for animal food. The Guidelines for Research Involving Recombinant DNA require the permanent marking of genetically engineered animals larger than rodents within 72 hr of birth. If their size does not permit permanent marking, their container should be marked.
- Immune deficiency as a potential concern in a genetically altered line should be addressed by containment and specific husbandry conditions to protect them from opportunistic microorganisms. Cesarian rederivation or continuous antimicrobial therapy may be necessary to control infectious diseases.
- Treatment should be considered as one option for welfare problems encountered in lines of genetically engineered animals. One common example is the administration of insulin to animals with type 1 diabetes mellitus.
- The limitation of gene _expression to certain tissues is another method to ensure welfare in altered animals. A genetic change that would cause morbidity or mortality when expressed in all tissues can be limited to certain tissues of interest, e.g. with the Cre-loxP recombination system. The use of inducible promoters is another method of limiting gene action by turning them on and off for specific time periods. Examples are tetracycline promoter (tetracycline administered with the food), doxycycline-inducible promoter (doxycycline via drinking water), the CYP1A1 promoter, and a metallothionein promoter.
- Another way to deal with animal welfare issues in genetically altered mice is the establishment of humane endpoints. Scoring systems can be used by the application of consistent criteria to many different situations.
IACUC Oversight of Welfare Issues:
The IACUC is responsible for reviewing and approving proposed animal studies. IACUC members may be faced with a variety of problems such as premature lethality, altered bodily functions, increased tumor production, decreased disease resistance, altered susceptibility to microorganisms. The first questions to be asked should concern what is known about the genetically altered line and what are the expected outcomes. The investigator should be asked to list the anticipated effects of the proposed manipulations and to describe the planned monitoring protocol for identifying problems.
One of the topics suggested for review of animal care and use protocols is "Criteria and process for timely intervention, removal of animals from a study, or euthanasia if painful or stressful outcomes are anticipated" (Guide for the Care and Use of Laboratory Animals, NCR 1996, p. 10).
If a new line will be established it may be difficult to predict outcomes accurately. However, there is value in anticipating the possible problems. The list of those may be revised as the study progresses. A vigilant surveillance system should be instituted and endpoints to be used for euthanasia of affected animals defined. When problems are encountered, the attending veterinarian and the investigative team should work together to identify ways to prevent or alleviate them.
1. Which of the research areas mentioned below benefit from the manipulation of the genome of animals?
a) gene discovery and test systems
b) disease models and gene therapy
c) xenotransplantation and life span extension
d) all of the above
e) none of the above
2. Which of the following statements is false ?
a) The research area "gene discovery" determines the structure and functions of various genes.
b) Proteomics is an area closely related to gene discovery and focuses on the therapy of diseases caused by proteoglycan deficiencies.
c) Xenotransplantation is the use of live, nonhuman animal cells, tissues, and organs in humans.
d) Gene therapy means treating disease conditions by altering the genome of somatic cells.
3. There are several reasons why the number of mice used in biomedical research increased sharply conflicting with the proposed reduction as a result of genetic engineering. Which of the following statements is true?
a) A relatively small number of animals is necessary to create each genetically modified line.
b) In point mutagenesis studies, where males get ENU administered up to six generations of breeding and screening are required to detect mutant progeny.
c) The increase in numbers of mice can be attributed to intensified research in areas that were previously hampered by a lack of adequate models.
d) Animals utilized to produce genetically modified lines deliver usable research data themselves.
4. Animal welfare issues are often unpredictable in newly created lines because of the variation in phenotype even if the same methodology and identical DNA constructs are used. What influences the phenotype of genetically engineered animals?
a) The number of copies of the inserted DNA construct and the location (particular chromosome or even different part of chromosome) of insertion.
b) The variation in regulatory sequences activated or inactivated.
c) The background strain of mouse used for the creation of a new line.
d) A and C are true.
e) All of the above are true.
5. There are different tools to identify, prevent, or limit animal welfare concerns in genetically altered animals. Which statement is false?
a) Databases should be searched to identify known characteristics of lines.
b) A clinical surveillance system should be established and containment measures taken to avoid escape of genetically altered animals.
c) The limitation of gene _expression to certain tissues and the use of inducible promoters have been proven ineffective.
d) Treatment and establishment of humane endpoints are ways to manage welfare concerns.
1d, 2b, 3c, 4e, 5c
Brazil-USA workshop: the future of animal research. ILAR 43 (2): 110.
Background and Introduction: Meeting objective was to strengthen ties between Brazilian and US scientists and students by exchanging perspectives about the use and care of laboratory animals. The following were common themes during the workshop. (1) Animal models are becoming increasingly important in research on prevention and treatment of diseases in humans, pets, farm animals, zoo animals, and research animals. (2) Genetically manipulated animals hold promise for understanding and treating human diseases but bring new challenges in regard to animal well-being. (3) Appropriate care and use of research animals require an emphasis on ethics pertaining to their well-being. (4) International cooperation and collaboration in research and in developing standards for animal care and use are essential for maximizing progress in improving quality of life for both people and animals.
Laws, Regulations, and Animal Welfare: Both countries have established strong traditions in animal welfare and have numerous laws, regulations, and policies to ensure appropriate care and use of animals. However, the complexity of laws, regulations, and policies has sometimes created regulatory burdens that impede scientific progress without affecting animal welfare. Education of researchers and staff on appropriate animal care and use is equally important. Training and education programs by organizations such as AALAS are critical. Site visits and recommendations by AAALAC International at accredited institutions are invaluable in improving animal care and use procedures and physical facilities.
Genetics and Cloning: The number of rodents used in research is increasing dramatically, and many institutions are struggling to keep up in terms of new housing facilities. Nonhuman primate (NHP) numbers, due to their close resemblance genetically and physiologically to humans, are also increasing because NHPs are serving as a link between rodent research and technologies used in human patients. Progress is being made in developing technology to produce transgenic monkeys. Animals are also being used for mutagenicity tests, primarily as it relates to the widespread use of pesticides. The US is the largest user of pesticides, with Brazil ranking third. Most mutagenicity tests measure somatic mutations but the significance of such mutations to cancer, health, and germline mutations is unclear. Brazilian law is based on prohibition of pesticides that demonstrated mutagenic effects whereas US law is based on risk assessment. The advent of transgenic crops can reduce pesticide use but may pose other risks such as causing food allergies in humans and transgenes being transported into wild varieties of crop species, possibly disrupting ecological balances. Current Brazilian law prohibits transgenic crops but US law is supportive of them in specific circumstances. New tests for mutagenicity and carcinogenicity are needed and will require the use of both lab animals and alternatives.
Research with Farm Animals and Conditions for Their Well-being: First scientific approach to livestock production was recorded in France in 1843. In 1893, the concept of farm animals as machines for mass food production was prevalent. Criticism of this later concept led to implementation of laws that provide animals with basic needs. However, the existence of such legislation as well as engineering standards does not necessarily ensure farm animal welfare. Performance standards are as important for farm animals as for lab animals and should be emphasized in laws, regulations, and practices. US is 2nd to China as the world's largest pork producer, and Brazil is the 8th largest producer. Swine are used for many human medical applications (source of insulin, thyroid hormone, heparin, hemoglobin, skin for burn treatments, cardiac valves, other biomaterials). Swine are used in research because some aspects of anatomy, physiology, and pathology resemble those of humans. Current research focus is on xenotransplantation. Transgenic swine with human genes have been created to reduce the risk of rejection of swine-to-human organ transplants, with gnotobiotic swine being developed to prevent transmission of swine viruses with xenotranplants. Current research uses swine-to-baboon transplant model to develop safe and effective technologies that can be applied to humans.
Research with Rodents: Chagas' disease is of increasing concern to the US due to migration from Latin America and transmission risks of the causative organism, Trypanosoma cruzi, by blood transfusion and organ transplantation. Different mouse strains exhibit varying degrees of infection resistance to T. cruzi and disease progression, and different T. cruzi strains vary in their ability to cause disease. Complex interactions between host and pathogen genetic makeup are critical factors in disease etiology. The mouse model will be key in investigating how these interactions affect immune function and for testing new treatments for T. cruzi infections. Research on this disease may help in understanding and preventing other parasitic diseases in humans and domestic animals.
Research with NHPs: Ironically, the biggest user of NHPs in research, the US, has no native NHP species. The growing demand for NHPs in research raises additional concerns concerning species conservation and capture policies. Because of a worldwide shortage of rhesus monkeys for research, significant efforts in the U.S. are under way to increase domestic production, especially of specific pathogen-free (SPF) rhesus monkeys. SPF monkeys are free of herpes B and of retroviruses that interfere with AIDS-related research. Because the mouse model of Chagas's disease does not closely mimic humans in some aspects of the immune response and does not develop human-like symptoms, NHPs are also used to study this disease. The baboon model is likely to be important in research on the epidemiology of T. cruzi infection, genetic and immunological bases of infection resistance and disease progression, and the development and testing of drugs to prevent or treat Chagas's disease.
Closing Session: Improving the quality of human life will continue to be dependent on the use of research animals. Degree of progress will in turn depend on appropriate care of laboratory animals and their judicious use to achieve experimental objectives.
1. A recently imported squirrel monkey is exhibiting signs of generalized edema and anemia. An EKG reveals a right bundle branch block. At necropsy, the myocardium contains cystic structures with oval shaped organisms. What is the most likely diagnosis?
a. Plasmodium knowlesi
b. Trypanosoma cruzi
c. Hepatocystis kochi
d. Theileria cellii
2. What 2 hemoflagellates infect NW NHP?
3. T. rangeli is basically nonpathogenic. Why?
4. In humans and some NW NHP what organ is often affected with T. cruzi and consequently fails?
5. To identify T. cruzi you would examine stained blood for the ________ form of the parasite.
6. How often does AAALAC International perform site visits on accredited institutions?
a. Every 6 months
c. Every 2 years
d. Every 3 years
7. What does AALAS and AAALAC International stand for?
8. To which of the 3 subfamilies (alpha, beta, or gamma herpesvirinae) of the Family Herpesviridae does Cercopithecine herpesvirus 1 (formerly known as Herpes B) belong?
9. Subcutaneous fibromatosis is associated with ____ retrovirus, whereas, retroperitoneal fibromatosis is associated with ______ retrovirus.
10. The primary receptor on the T-helper cell for SIV and HIV is ______.
11. T/F. SRV-1 and SRV-2 are the primary causes for SAIDs cases.
2. Trypanosoma cruzi & T. rangeli
3. It's found only in blood and has no tissue stage or intracellular multiplication stage
4. Heart affected in humans, right bundle branch block also apical aneurysms
7. American Association of Laboratory Animal Science (AALAS); Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC) International.
9. SRV-1/ SRV-2 Type D