ANAT2341 Lab 4 2013
Mouse Models of Human Genetic Disease
Objectives of this laboratory
- Short answer/multiple choice test of last week's lecture material ('mesoderm' and 'ectoderm, early neural and neural crest')
- Tutorial on the technologies available to researchers to study gene function in mice.
- Provide time for groupwork and allow groups to ask questions of lecturing staff.
Tutorial
In the tutorial I will explain why and how researchers are using the mouse as a model for human genetic disease. I will give an overview about the various methods to study gene and protein expression in the developing mouse embryo, and how researchers manipulate gene function in mice in vivo. This tutorial will greatly facilitate your understanding of the research articles that you will read for your group projects. A PDF file with the powerpoint show can be found here: File:ANAT2341 - Lab 4 - Mouse Models of Human Disease.pdf
Learning Resources
- 'Molecular Biology' by Turner. Section T: 'Functional Genomics and New Technologies'. eBook in UNSW library: [1]
- 'Developmental Biolgy' by Scott Gilbert. Chapter 4: The Genetic Core of Development. [2]
- Review Article: 'Knock Out, Knock In, Knock Down — Genetically Manipulated Mice and the Nobel Prize' by John P. Manis, M.D. File:Classic KO technology.pdf
- Review article: 'Animal Transgenesis' by Miguel A. Gama Sosa, Rita De Gasperi, Gregory A. Elder. File:BSF transgenesis review.pdf
Why do researchers use the mouse as a model system for human disease?
- Small, cheap to house and feed, breed quickly.
- Embryology resembles that of humans.
- Genetically similar to humans.
- Fully sequenced genome.
- Amenable to genetic manipulation.
Gene and protein expression technologies
Our genetic material, the DNA, is localized in the nucleus, and encodes proteins, which carry out a diverse range of functions in- and outside the cells. DNA is first transcribed into messenger RNA, which is then translated into proteins in the cytoplasm. The first step to get insight into gene or protein function, is by knowing where it is localized. Numerous technologies have been developed that investigate the location of RNA and protein in tissues and cells. I will shortly discuss the following technologies in the lab:
Protein detection:
- Protein gels and Western blotting
- Proteomics
- Immunodetection in tissue sections
RNA detection:
- Reverse transcriptase polymerase chain reaction (RT PCR)
- RNA gels and Northern Blotting
- Quantitative real time RT PCR
- In situ hybridization (sections/whole embryos)
- Transcriptome profiling using microarrays
Gene function analysis in mice
There are two approaches to study gene function in an organism: forward and reverse genetics.
Forward genetics is the classic way to study gene function, and it is still commonly used by researchers today. With this type of approach the abnormal phenotype of a mutant organism is studied to get an insight into the function of the mutated gene, of which the nature intitally is unknown. This comprises studies of for instance naturally occurring mutants and random mutagenesis screens. Positional cloning approaches using known marker alleles will allow reserachers to pin down the affected chromosomal locus and to eventually identify the nature of the mutation.
Reverse genetics approaches start of with the DNA sequence of a particular gene with unknown function. Modern approaches allow targeted manipulation of the genome of an organism, and generation of mutations in a gene of choice. This will result in phenotypic abnormalities that are informative about the role of the gene in that organism.
The mouse is an excellent animal model to study gene function through reverse genetics, though forward genetics screens are also still commonly done. I will discuss the following common approaches to study gene function in mice in the lab:
- Gain-of-function transgenesis
- Loss-of-function transgenesis
- Conventional gene loss-of-function technology
- Conditional gene loss-of-function technology
- Random mutagenesis screens
