ANAT2341 Lab 5 2013

From Embryology

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.

  1. 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.
  2. 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
  3. 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

What's Wrong with My Mouse?

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 Sometimes cell lines are made from genetically modified mice in order to conduct specific tests in cell culture conditions. For example - mouse embryonic fibroblasts are frequently used for a variety of purposes and keratinocyte cultures are often made from skin samples.

Phenotype analysis - Developmental

One of the great advantages of mouse models is the ability to study pathology at any time point throughout development with much less ethical constraint. Tissue can be collected and snap frozen immediately (not during autopsies conducted many hours later) making it feasible to do a variety of different techniques. For example: high quality histology for immunohistochemistry and immunofluorescence or electron microscopy; collection of tissues for protein, RNA or DNA extraction or collection of cells and tissues for cell culture, cell sorting or organ culture.

One main aim is to pinpoint the earliest time point at which things seem to go wrong. Clearly, one would expect things to go wrong in the cell type in which the gene is normally expressed - hence the importance of determining temporal and spatial patterns of expression.

Then we can ask questions about why the cells do not behave normally.

Do the cells proliferate properly - dysregulation of the cell cycle control machinery? Cancer? e.g. Gorlin syndrome/Basal Cell Nevus Syndrome

Is there a defect of one of the cell lineages? - neural crest derived cells affected in Treacher-Collins and Waardenburg Syndrome type 4

Is the gene regulating the expression of other genes and if so, what are those gene targets? DLX5 in Split-hand/foot malformation is a homeobox gene T-Box genes - TBX5, TBX1, T, SOX9, PAX6, RUNX2.

Are cell signalling mechanisms involved? - Androgen Receptor (AR) and Endothelin Receptor Type B (ENDRB)

What is the cellular consequence of loss of gene function?

What is the molecular consequence of loss of gene function?

Phenotype analysis - Behaviour

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

  1. Sensory capabilities - hearing, vision, touch, smell, pain etc
  2. Motor functions - General motor impulse during the 24hr cycle, motor coordination control, gait and muscle strength
  3. Learning and memory
  4. Feeding and drinking
  5. Reproductive behaviour
  6. Social behaviour
  7. Emotional behaviour
  8. Reward (e.g. self-administration of addictive drugs)

Molecular analysis

Once phenotypes have been established and attributed to specific tissues or cell types, it often desirable to probe the molecular cause by conducting biochemical or molecular analyses. This usually involves a direct comparison between protein or nucleic acid samples extracted from the target tissue of a set of genetically modified mice compared with an equal number of samples from control wild-type siblings.

Some examples include:

  1. Gene expression profiling using microarray or RNA-seq strategies
  2. Multiplex protein abundance measurements using 2D gel electrophoresis
  3. Direct candidate gene expression analysis using quantitative RT-PCR
  4. Direct candidate protein abundance measurements using Western blotting
  5. Analysis of pathway activation using markers of protein phosphrylation

Epigenetics tests

Recently there has been a dramatic expansion in interest in epigenetic marks as mediators of gene expression control. Therefore, modern molecular strategies often include tests that probe candidate tissues for epigenetic markers such as

  1. DNA methylation
  2. Histone methylation
  3. Histone acetylation
  4. Nucleosome depletion
  5. Non-coding RNA

and genes of interest that are involved in transcriptional regulation may involve experiments that are designed to examine direct interaction of the protein under study interacting with target sites on genomic DNA. ChIP (Chromatin immunoprecipitation) techniques.


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Cite this page: Hill, M.A. (2020, February 22) Embryology ANAT2341 Lab 5 2013. Retrieved from https://embryology.med.unsw.edu.au/embryology/index.php/ANAT2341_Lab_5_2013

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© Dr Mark Hill 2020, UNSW Embryology ISBN: 978 0 7334 2609 4 - UNSW CRICOS Provider Code No. 00098G