page_title: Reverse genetics
section_title: Directed deletions and point mutations
hierarchical_section_title: Reverse genetics / Techniques used / Directed deletions and point mutations
caption_attribution_description: English: Deviating phenotypes induced in gene-disruption library transformants. Physcomitrella wild-type and transformed plants were grown on minimal Knop medium to induce differentiation and development of gametophores. For each plant, an overview (upper row, scale bar corresponds to 1 mm) and a close-up (bottom row, scale bar equals 0.5 mm) is shown. A, Haploid wild-type moss plant completely covered with leafy gametophores and close-up of wild-type leaf. B-D, Different Mutants.
context_page_description: Reverse genetics is a method in molecular genetics that is used to help understand the function of a gene by analysing the phenotypic effects caused by genetically engineering specific nucleic acid sequences within the gene. The process proceeds in the opposite direction to forward genetic screens of classical genetics. While forward genetics seeks to find the genetic basis of a phenotype or trait, reverse genetics seeks to find what phenotypes are controlled by particular genetic sequences.
  Automated DNA sequencing generates large volumes of genomic sequence data relatively rapidly. Many genetic sequences are discovered in advance of other, less easily obtained, biological information. Reverse genetics attempts to connect a given genetic sequence with specific effects on the organism.
context_section_description: Site-directed mutagenesis is a sophisticated technique that can either change regulatory regions in the promoter of a gene or make subtle codon changes in the open reading frame to identify important amino residues for protein function.
  Alternatively, the technique can be used to create null alleles so that the gene is not functional.  For example, deletion of a gene by gene targeting (gene knockout) can be done in some organisms, such as yeast, mice and moss. Unique among plants, in Physcomitrella patens, gene knockout via homologous recombination to create knockout moss (see figure) is nearly as efficient as in yeast. In the case of the yeast model system directed deletions have been created in every non-essential gene in the yeast genome. In the case of the plant model system huge mutant libraries have been created based on gene disruption constructs. In gene knock-in, the endogenous exon is replaced by an altered sequence of interest.
  In some cases conditional alleles can be used so that the gene has normal function until the conditional allele is activated.  This might entail 'knocking in' recombinase sites (such as lox or frt sites) that will cause a deletion at the gene of interest when a specific recombinase (such as CRE, FLP) is induced.  Cre or Flp recombinases can be induced with chemical treatments, heat shock treatments or be restricted to a specific subset of tissues.
  Another technique that can be used is TILLING. This is a method that combines a standard and efficient technique of mutagenesis with a chemical mutagen such as ethyl methanesulfonate (EMS) with a sensitive DNA-screening technique that identifies point mutations in a target gene.
caption: Wild-type Physcomitrella patens (A) and Knockout mosses (B-D): Deviating phenotypes induced in gene-disruption library transformants. Physcomitrella wild-type and transformed plants were grown on minimal Knop medium to induce differentiation and development of gametophores. For each plant, an overview (upper row; scale bar corresponds to 1 mm) and a close-up (bottom row; scale bar equals 0.5 mm) are shown. A: Haploid wild-type moss plant completely covered with leafy gametophores and close-up of wild-type leaf. B–D: Different mutants.[2]
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