Revision as of 15:20, 26 July 2013

Understanding Mechanisms Using Mouse Models

Genetic Background

One of the major advantages of using mouse models is that they are generally made on an inbred genetic background. This means that all mice are genetically identical except for the genetic modification that is under study. These are referred to as congenic mouse lines. This lack of genetic variability limits the background variance and allows phenotypic changes to be attributed directly to genotype. The most common mouse line in use is called C57BL/6

The test of survival - Mendelian ratios

Some genetic modifications may threaten the survival of mice during development or cause pre-weaning death due a "failure to thrive". The average litter size for the C57BL/6 strain is approximately 6 After a few litters it may become apparent that the expected Mendelian ratios are not being met. This can be tested statistically using the Chi Square test.

For example: A mouse that is hemizygous for a transgene insertion crossed with a wild type mouse would be expected to produce 50% hemizygous transgenic offspring and 50% wild type offspring. A mouse that has a heterozygous deletion in an endogenous gene crossed with another heterozygous mutant would be expected to produce wild types: heterozygotes amd homozygous mutants at a ratio of 1:2:1

Examining expression of the gene of interest

One of the main clues as to where to expect phenotypic consequences is a good knowledge of the expression pattern of the gene of interest. This can be studied from a spatial point of view - i.e. which organs/tissues/cells express the gene of interest, or from a temporal point of view - when does expression begin in these locations (during development?) and when does it cease? There are several ways of achieving this aim.

Using anti-gene-of-interest antibodies on histological tissue sections to determine where the encoded protein is located. This can be technically challenging as it requires a good antibody, abundant levels of protein and good access of the antibody to the location.
RNA In-situ hybridization (ISH). This method is more standardized and predictable but there is still a requirement for reasonably abundant levels of messenger RNA. Several databases are available that curate RNA ISH experiments and most genes in the genome now have at least some data using this technique. GenePaint Website - RNA ISH on sections of developing mouse embryos Allen Brain Atlas Website - RNA ISH on sections of mouse brain
Knock-in of reporter into endogenous gene of interest. A common strategy when making a mouse knockout is to also knock-in a recombinant DNA sequence encoding a reporter into the endogenous gene locus. Some examples of reporters that are commonly used include the E.coli LacZ gene, which can be detected using a simple histochemical technique that leaves a blue signal wherever the endogenous gene is normally expressed. GFP - A fluorescent tag based on a jellyfish protein that will glow green when stimulated by UV. Human placental alkaline phosphatase - another histochemical marker.

Phenotype analysis - Physical

Body weight

A simple test that says a lot about impairments in growth and development, is minimally invasive (at postnatal stages) and can be collected throughout the lifespan of the mouse to look at ability to meet growth milestones.

Histology

Standard pathological techniques that are used for human subjects are also available here to look for evidence of similar pathology to the human condition. However, in the mouse, there are less ethical restrictions and therefore any tissue can be accessed at any age. Therefore, more detailed analyses can be conducted with larger sample sizes and less genetic background variation.

Organ/tissue/cell specific tests

Similarly, with appropriate ethics approval, organs and tissues can be extracted for biochemical analysis, extraction of nucleic acids, proteins, etc

Behavioural tests

To test functional abilities, a range of test equipment and protocols have been designed to probe functions such as:

Sensory capabilities - hearing, vision, touch, smell, pain etc
Motor functions - General motor

Molecular analysis

Glossary Links

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Cite this page: Hill, M.A. (2024, April 23) Embryology ANAT2341 Lab 5 2013. Retrieved from https://embryology.med.unsw.edu.au/embryology/index.php/ANAT2341_Lab_5_2013

What Links Here?

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 The average litter size for the C57BL/6 strain is approximately 6
 After a few litters it may become apparent that the expected Mendelian ratios are not being met. This can be tested statistically using the Chi Square test.
 For example:
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-==Phenotype analysis==
+==Phenotype analysis - Physical==
 ===Body weight===
+A simple test that says a lot about impairments in growth and development, is minimally invasive (at postnatal stages) and can be collected throughout the lifespan of the mouse to look at ability to meet growth milestones.
 ===Histology===
-===Organ specific tests===
+Standard pathological techniques that are used for human subjects are also available here to look for evidence of similar pathology to the human condition. However, in the mouse, there are less ethical restrictions and therefore any tissue can be accessed at any age. Therefore, more detailed analyses can be conducted with larger sample sizes and less genetic background variation.
+===Organ/tissue/cell specific tests===
+Similarly, with appropriate ethics approval, organs and tissues can be extracted for biochemical analysis, extraction of nucleic acids, proteins, etc
 ===Behavioural tests===
+To test functional abilities, a range of test equipment and protocols have been designed to probe functions such as:
+#Sensory capabilities - hearing, vision, touch, smell, pain etc
+#Motor functions - General motor
 ==Molecular analysis==

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