Multicellular living organisms and unmodified parts thereof and – Nonhuman animal – Transgenic nonhuman animal
Reexamination Certificate
1999-08-12
2003-02-11
Yucel, Remy (Department: 1632)
Multicellular living organisms and unmodified parts thereof and
Nonhuman animal
Transgenic nonhuman animal
C800S021000, C435S320100, C435S455000, C435S473000, C532S001000
Reexamination Certificate
active
06518481
ABSTRACT:
INTRODUCTION
1. Field of the Invention
The field of the invention is markers for identifying transgenic animals.
2. Background of the Invention
Genetic manipulation of insects and other arthropods is a highly desirable goal for the development of better control strategies to fight agricultural pests and disease vector species. Transposon-based transformation techniques had been available for Drosophila, but only recently did the discovery of new transposons enable this approach in other insects (O'Brochta & Atkinson, 1996, Insect Biochem. Molec. Biol. 26, 739-53), i.e medflies (Loukeris et al., 1995, Science 270, 2002-5; Handler et al., 1998, PNAS 95, 7520-5) and mosquitoes (Coates et al., 1998, PNAS 95, 3748-51; Jasinskiene et al., 1998, PNAS 95, 3743-7). However, a major obstacle in the use of these transposons has been the difficulty to obtain marker genes that allow easy and reliable identification of transgenic animals. In fact, a main reason why germline transformation experiments have not been carried out routinely so far in non-dipteran insects, is the lack of specific markers to follow gene transfer (DeVault et al., 1996, Genome Research 6, 571-9). Here we present a novel marker system broadly suitable for eye-bearing animals.
In combination with a set of promiscuous vectors, our system permits the study of biologically relevant questions in almost any species, not only in established model organisms. Since the very same system can be used in a series of different organisms, comparative biological and functional evolutionary studies are facilitated, providing a vital tool for the emerging field of evolutionary developmental biology. Furthermore, expression in the eyes allows visualization of the signal in animals with non-transparent cuticle, and transgenic animals can be identified as larvae, pupae and adults. Together with the fact that the system can be applied to competitive wild type strains rather than potentially labile mutant lines, makes the system particularly applicable to pest management programs.
SUMMARY OF THE INVENTION
The subject methods generally comprise (a) introducing into a genome of an animal a genetic construct comprising a transcriptional regulatory element operably linked to a heterologous marker gene encoding a marker, wherein the element drives expression of the marker across genera transgenic in the construct sufficient to visually detect the marker in photoreceptive cells or organs, and (b) selecting for transgenesis by visually detecting the marker in a photoreceptive cell or organ of the animal. In particular embodiments, the construct comprises a vector, such as transposon or retrovirus, particularly a polytropic vector. The construct may integrate into the genome by homologous or non-homologous recombination. In particular embodiments, the transcriptional regulatory element comprises a binding site selected from a Pax-6 binding site, a Glass binding site, etc., particularly a plurality of P3 sites, and the marker is a fluorescent protein, particularly a green fluorescent protein or variant thereof.
The subject compositions include polytropic vectors functional in nondipteran species and comprising a transcriptional regulatory element operably linked to a heterologous marker gene encoding a marker, wherein the element drives expression of the marker across genera transgenic in the construct sufficient to visually detect the marker in photoreceptive cells or organs, particularly wherein the marker is the only visually detectable indicator of transgenesis encoded by the vector. The invention also provides cells and animals transgenic in the subject constructs and/or made by the subject methods.
DETAILED DESCRIPTION OF PARTICULAR EMBODIMENTS OF THE INVENTION
The following descriptions of particular embodiments and examples are offered by way of illustration and not by way of limitation. Unless contraindicated or noted otherwise, in these descriptions and throughout this specification, the terms “a” and “an” mean one or more, the term “or” means and/or and polynucleotide sequences are understood to encompass opposite strands as well as alternative backbones. The subject methods and applications are applicable to a wide variety of photoreceptor cell or organ bearing animals. By photoreceptive cells or organs is meant any light sensing cell or organ of an animal and include cells such as simple pigmented light sensitive cells or retinular cells and structures such as ocelli also called simple eyes or eye spots like the direct or inverted pigment cups of many worms, structures such as compound eyes found in many arthropods, structures such as complex eyes or camera eyes of cephalopod molluscs and vertebrates. Unless otherwise noted, the term eye is used herein to collectively refer to these various light sensing cells or organs. The suitability of any particular photoreceptor cell or organ bearing animal is readily determined empirically, using conventional genetic transformation procedures and screening procedures, as exemplified below. Genera demonstrating transgenesis according to the disclosed methods include vertebrates, particularly mammals, fish and birds, and non-arthropod and arthropod invertebrates, such as Crustacea, Chelicerata and Insecta, such as Diptera such as flies and mosquitoes, and non-dipteran insects, such as Lepidoptera, Hymenoptera, Coleoptera, Neuroptera, Hemiptera, Isoptera, Dictyoptera, and Orthoptera.
The subject methods employ a transcriptional regulatory element operably linked to a heterologous marker gene encoding a marker, wherein the element drives expression of the marker across genera transgenic in the construct sufficient to visually detect the marker in photoreceptive cells or organs. By drives expression across genera is meant that the element is capable of promoting gene expression in a plurality of genera, preferably including a non-dipteran insect, more preferably including a non-insect arthropod. Preferred elements are functional in a plurality of taxonomic (Zoological Record, BIOS UK, 1999) families, preferably a plurality of orders, more preferably a plurality of classes, more preferably a plurality of phyla. In particular embodiments, the element is functional in at least the families Drosophilidae, Calliphoridae and Culicidae, preferably in the orders Diptera, Lepidoptera and Coleoptera, more preferably in the classes Insecta, Malocostraca and Chelicerata, even more preferably across the phyla Arthropoda, Mollusca and Chordata.
To drive marker expression in a series of diverged organisms requires a promoter which is active in a wide range of species. Furthermore, to avoid problems with low expression and the interference of autofluorescence, a regional specific promoter is preferable over a constitutively active one. A wide variety of regulatory elements may be employed, so long as they meet the requisite functional limitations. These may be natural promoter elements, naturally driving gene expression in photoreceptive cells or organs, elements derived from such natural promoter elements by mutational selection or consensus sequences, synthetic elements derived by iterative selection process, e.g. SELEX procedures, etc. In a particular embodiment, the element comprises a binding site selected from a Pax-6, a Pax-6 like binding site such as a twin-of-eyeless (TOY) binding site, a Glass binding site, etc. In more particular embodiments, the element comprises a Pax-6 Paired Domain or Homeodomain binding site, more particularly a P3 site, wherein the P3 site comprises the sequence: TAATYNRATTA (SEQ ID NO:01), wherein Y=C or T; R=G or A; N=any nucleotide (Wilson et al., 1993, Genes Dev 7, 2120-34; Czerny and Busslinger, 1995, Mol Cell Biol 15, 2858-71). Tables 1-6 provide other exemplary transcriptional regulatory element binding sites functional in the subject methods. Pax-6 binding sites are of particular interest due to the evolutionary conserved role Pax-6-homologs play in eye development across different phyla (Callaerts et al., 1997, Annu Rev Neurosci 20, 483-532).
TABL
Berghammer Andreas J.
Klingler Martin
Wimmer Ernst A.
Exelixis Inc.
Nguyen Quang
Osman Richard Aron
Yucel Remy
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