8 Results
for term "sites"
- The Genomic and Genetic Toolbox of the Teleost Medaka (Oryzias latipes)...of such approaches is that in most cases these mutations can be identied by locating the transgene insertion sites within the genome. In medaka, gene trap approaches with several transposons have been successfully employed (Sano et al. 2009; Froschauer et al. 2012). The transposase Frog Prince has been used ~~~
- Brainbow: New Resources and Emerging Biological Applications for Multicolor Genetic Labeling and Analysis...-like strategies are now used, utilizing recombinase-mediated DNA excision or DNA inversion (Figure 1A). In DNA excision-based Brainbow (e.g., Brainbow 1.0), three separate FPs are arranged sequentially in the transgene along with two pairs of Cre recombinase recognition sites (Lox sites) that ank the rst ~~~
- Genetic and Genomic Toolbox of Zea mays...engineered with lox recombination sites so that transgenes can be inserted and manipulated without affecting the A chromosomes (Yu et al. 2007). In addition to these traditional genetic resources, there is a large collection of uorescent protein marker lines (Mohanty et al. 2009; Wu et al. 2013). Among ~~~
- Tetrahymena as a Unicellular Model Eukaryote: Genetic and Genomic Tools...anlagen) develops during conjugation (Figure 3, H and I) these 10 chromosomes are fragmented at specic sites called chromosomal breakage sequences (CBSs) and they lose some internal sequences (Yao et al. 1987), collectively referred to as internal eliminated sequences (IESs). Fragmentation and DNA ~~~
- The Expanding Genetic Toolbox of the Wasp Nasonia vitripennis and Its Relatives.... Literature Cited Abdel-Latief, M., L. A. Garbe, M. Koch, and J. Ruther, 2008 An epoxide hydrolase involved in the biosynthesis of an insect sex attractant and its use to localize the production site. Proc. Natl. Acad. Sci. USA 105: 89148919. Akbari, O. S., I. Antoshechkin, B. A. Hay, and P. M. Ferree, 2013 ~~~
Figure 1Principles of Brainbow labeling. (A) Cre recombinase can perform excision or inversion of DNA flanked by Lox sites (triangles), depending on the orientation of the Lox sites. Different lox sites such as lox2272 (black triangle), loxP (white triangle), and loxN (gray triangle) function identically but are incompatible with each other. (B) Excision-based Brainbow. Fluorescent proteins (FPs) are flanked by two pairs of mutually incompatible Lox sites. In the absence of recombination, RFP is expressed. Recombination results in excision expression of either CFP (event 1) or YFP (event 2). (C) Inversion-based Brainbow. FP expression can be changed between RFP and CFP by DNA inversion. (D) In Brainbow 2.1, DNA excision leads to selection of either the GFP/YFP pair or the RFP/CFP pair. DNA inversion then decides which FP of the pair is expressed. Brainbow AAV works similarly. (E) For each copy of Brainbow, only the first FP in the array is expressed. Therefore in a cell population with a single Brainbow transgene, cells can be RFP+ (no recombination, i.e., “default”), CFP+, or YFP+. (F) When multiple copies of Brainbow are present in a cell, each copy recombines independently. Three copies of Brainbow can generate 10 distinct colors and more copies will generate even greater color diversity. (G) Combinatorial multicolor labeling can also be achieved by using multiple vectors, each carrying a single FP. As the expression of each FP is stochastic, the color profile within each cell is different. B and F are modified from Pan et al. (2013).
Figure 6Current and emerging applications. (A) A cerebellar folium from the Brainbow mouse line H was imaged using confocal microscopy. Three-dimensional volume (160 μm2 × 65 μm) indicated in the box was segmented using semiautomated methods and reconstructed digitally, as shown in B. (B) Digital reconstruction of 341 axons and 93 granule neurons from volume marked in A. A and B are modified from Livet et al. (2007). (C) Multicolor cells can be followed over time in living tissue and then sorted by color (e.g., FACS) for sequencing or gene expression analysis. (D) In this schematized construct, a particular gene (gene A*) is coexpressed with YFP (following excision at loxP site 2). In the resulting cell population at right, only cells expressing any level of YFP will also express the gene of interest.
Figure 4Gene expression and cell lineage analysis in medaka. (A) Whole-mount in situ hybridization to specifically detect retinal homeobox gene Rx2 mRNA in the retina. Rx2 (red) is expressed in photoreceptor cells (PR), stem cells (RCS) in the ciliary marginal zone of the retina, and Mueller glia cells (MGC). L, lens. Glutamine synthase protein in Mueller glia cells is codetected by antibody staining (white), indicating Rx2 expression in Mueller glia cells (red dots inside white MGCs). (B) Recombination scheme: the GFP reporter gene is present in the PhiC31 landing site. The regulatory DNA to drive GFP reporter gene expression is provided with a targeting vector. PhiC31 integrase activity leads to the recombination of the regulatory DNA into the GFP reporter gene cassette in the genome. In vivo imaging of neurobal GFP expression from a brain-specific enhancer within a PhiC31 landing site. Note autofluorescent pigment cells (red). (C) In vivo analysis of 8-dpf Gaudí fish. Stochastic recombination of a brainbow cassette by heatshock-activatable Cre recombinase results in a differential labeling of cells in the entire body. A 3D representation of the somites is shown. (D) A whole-mount BrdU incorporation assay in a 10-dpf hatchling reveals proliferating cells (yellow) in the body. Nuclei are counterstained with Dapi (blue).