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16.2:

Test genetici

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Biologia Molecolare
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JoVE Core Biologia Molecolare
Genetic Screens

Lingue

Condividere

Genetic screens are analytical tools for studying the correlation between the genotype and the phenotype of an organism. They are broadly classified into forward genetic screens and reverse genetic screens. Forward screens involve identifying the genes responsible for an observed characteristic, such as petal color, thus using a known phenotype to study an unknown genotype. These screens usually involve the genome of an organism being randomly mutated to induce a change in phenotype. Next, the mutant organisms are bred until genes are homozygous, to ensure expression of recessive phenotypes. Organisms are then screened for traits of interest. For example, to identify genes responsible for morphological defects in zebrafish embryos, sperm cells of adult males are mutated with a chemical, ethylnitrosourea, and then crossed with a wild type female. The resulting F1 males are crossed again with a wild type female and dominant mutations observed in the F2 progeny. F2 individuals are then inbred to identify recessive gene mutants in F3 individuals.    Genes responsible for the altered phenotype can then be determined by different methods. In one technique, genomes of multiple mutant and wild type organisms are sequenced.  A region that is similar in all mutants, but not the wild types, helps in locating the mutated gene. Additionally, forward genetic screens can be used to identify mutations that enhance or suppress the severity of a phenotype in organisms. These are known as modifier screens. In contrast, reverse genetic screens go from genotype to phenotype and examine the phenotype resulting from mutating a specific gene with an unknown function. For example, consider the genes for molecular chaperones in Drosophila.  Molecular chaperones are proteins essential for the folding of other proteins in different parts of the organism. Targeted knockdown of different chaperone genes in Drosophila eyes can result in observable morphological defects, thereby helping to identify which of these chaperones are essential for eye development. Alternatively, expression screening is another reverse genetics method. Here, a gene with an unknown function is expressed in a different organism, and changes in the phenotype are observed in the host to determine gene function.

16.2:

Test genetici

Genetic screens are tools used to identify genes and mutations responsible for phenotypes of interest. Genetic screens help identify individuals or a group of people at risk of developing  genetic diseases and help them with early intervention, targeted therapy, and reproductive options.

Forward genetic screens

Forward or “classical” genetic screens involve creating random mutations in an organism’s DNA using radiation, mutagens, or insertion of additional bases, which result in visible changes in the phenotype. The mutant is inbred to obtain progenies that are homozygous for the mutation. The mutation and its associated phenotype are identified, and gene locus on the chromosome are mapped.

Reverse genetic screens

Reverse genetic screens involve disruption of known genes, followed by screening for mutant phenotypes resulting from these manipulations. Expression screening is a type of reverse genetic screen that involves vector libraries containing the protein-coding sequences of various genes extracted from the genome of an organism or environmental samples. These screens help in the identification of novel proteins. For example, in zebrafish, using reverse genetic screening, the genes involved in early development are identified.

Applications

Genetic screens have several applications, such as identifying protein interactions, characterizing gene-drug interactions, and understanding the cause of diseases. For example, a mutant yeast library generated using large-scale random mutagenesis with transposon insertions can be grown in the presence of a drug. The effect of the drug on each mutant can be analyzed using PCR followed by microarrays or sequencing analysis. Similarly, screens can be used for the analysis of genes and molecular networks disrupted in human disease. For example, genes involved in neurodegenerative diseases can be identified by culturing neurons in the presence of RNA-encoding viruses to knock down the expression of different target genes. Cells can then be immunostained and analyzed to detect physical abnormalities and identify genes responsible.

Suggested Reading

  1. St Johnston, D. The art and design of genetic screens: Drosophila melanogaster. Nat Rev Genet 3, 176–188 (2002). https://doi.org/10.1038/nrg751
  2. Fuentes, R., Letelier, J., Tajer, B., Valdivia, L. E., & Mullins, M. C. (2018). Fishing forward and reverse: Advances in zebrafish phenomics. Mechanisms of development, 154, 296–308. https://doi.org/10.1016/j.mod.2018.08.007