Multicellular living organisms and unmodified parts thereof and – Method of making a transgenic nonhuman animal
Reexamination Certificate
1998-11-13
2001-06-19
Martin, Jill D. (Department: 1632)
Multicellular living organisms and unmodified parts thereof and
Method of making a transgenic nonhuman animal
C800S022000, C800S025000, C800S018000, C435S455000, C435S320100, C435S325000, C435S069100
Reexamination Certificate
active
06248934
ABSTRACT:
INTRODUCTION
1. Field of the Invention
The field of the invention is gene trap vectors.
2. Background
Previous analyses of mutations in axon guidance molecules in vertebrates have been hampered by the lack of histochemical markers to selectively study particular axons among the mass of the neuropil (e.g. Baier et al., 1996; Serafini, et al., 1996). Analysis of the projections of neurons has been carried out using time-consuming dye-fill techniques, or existing histochemical markers which are limited mainly to widely-expressed antigens, such as TAG-1, which stains all commissural axons. Such labels do not permit one to visualize subpopulations of axons or to distinguish partially penetrant effects on many axons from fully penetrant effects on few axons.
Gene trapping in mouse embryonic stem cells offers a versatile and cost-effective approach to creating large numbers of insertional mutations that are immediately accessible to molecular characterization (Gossler et al., 1989; Friedrich and Soriano, 1991; Skarnes, Auerbach & Joyner, 1992; von Melchner et al., 1992). Activation of the reporter gene depends upon its insertion within an active transcription unit, thereby creating a mutation in the gene at the site of integration. The trapped gene can be sequenced rapidly using PCR-based methods (Townley et al., 1997) and the expression profile of the target gene can be readily determined by monitoring reporter gene activity in ES cell-derived chimeric embryos (Wurst et al, 1995) and/or in ES cell cultures following in vitro differentiation (Forrester et al., 1996). Thus, the advantage of the gene trap approach resides in the ability to pre-select insertional mutations of interest based on sequence and expression information prior to germline transmission and phenotype analysis.
A modification of the gene trap approach, the secretory trap (Skarnes, et al., 1995), allows one to recover selectively insertions into transmembrane or secreted protein-encoding genes, thus greatly enriching for mutations in genes expected to include axon guidance ligands and receptors. The secretory trap method has already been highly successful in identifying insertions into transmembrane or secreted genes expressed in the nervous system (Skarnes et al. 1995; Townley et al., 1997). In particular, LAR, netrin-1, neuropilin-2 and Semaphorin C, have already been implicated in axon guidance in mice and/or in other systems (reviewed in Tessier-Lavigne and Goodman, 1996).
We have developed a further modification of the gene trap approach to screen efficiently for genes involved in establishing correct patterns of neuronal connectivity in the mouse. This system utilizes a vector comprising both a gene trap module, in this case a secretory trap, and an axonal reporter to mark the axons of only those cells that normally express the trapped gene. Using this vector the trajectories of labeled axons can be compared between homozygous mutant and heterozygous mice to determine whether the trapped gene is required cell-autonomously for guidance of those axons. This method will also reveal whether the gene is involved in other aspects of nervous system development such as elaboration or pruning of axonal arbors or neuronal survival.
Relevant Literaure
A recent issue of the journal Developmental Dynamics was devoted to gene trap technology and included relevant articles by Voss et al. (1998) Dev Dynamic 212:171-180; Xiong et al. (1998) Dev Dynamic 212:181-197; and Stoykova et al. (1998) Dev Dynamic 212:198-213.
Ito K, Sass H, Urban J, Hofbauer A, Schneuwly S, (October 1997) Cell Tissue Res 290 (1): 1-10, describe the use of GAL4-responsive UAS-tau as a tool for studying the anatomy and development of the Drosophila central nervous system. Callahan C A, Thomas J B (Jun. 21, 1994) Proc Natl Acad Sci USA 91(13):5972-6 describe the use of a tau-beta-gal enhancer-trap transposon to study Drosophila neural development. Mombaerts et al. (1996) Cell 87, 675-686 describes the use of targeted mutagenesis to visualize an olfactory sensory map.
Relevant patent documents include Plasterk et al. (1997) WO97/29202, Mark et al. (1998) WO98/23933, Smith et al. (1994) WO94/24301, Ruley (1997) U.S. Pat. No. 5,627,058 and Skarnes (1998) U.S. Pat. Nos. 5,767,336 and 5,789,653.
SUMMARY OF THE INVENTION
The invention provides methods and compositions for identifying vertebrate genes and/or gene function required for correct wiring of the nervous system. The compositions include gene trap vectors comprising a polynucleotide comprising promoterless selectable marker and axon reporter encoding sequences, whereupon transfer into an embryonic stem cell and integration of the polynucleotide into a gene of the cell, the cell expresses the selectable marker and the axon reporter under the transcriptional control of the gene. In particular embodiments, the axon reporter coding sequence is operatively joined to the selectable marker by an internal ribosome-entry site, such that upon transfer into the cell, expression of the selectable marker and the reporter is detectable upon integration into an active transcription unit.
The invention provides several methods of expressing targeted gene products in neurons. In one embodiment the method comprises the step of transferring into a vertebrate embryonic stem cell vectors comprising a polynucleotide comprising promoterless selectable marker and axon reporter encoding sequences, under conditions whereby the polynucleotide integrates into the gene and the cell or a progeny of the cell expresses the selectable marker and the axon reporter under the transcriptional control of the gene.
In another embodiment, the method comprises the steps of (1) transferring into the embryonic stem cell a gene trap vector comprising a polynucleotide comprising promoterless selectable marker and axon reporter encoding sequences under conditions whereby the polynucleotide integrates into the gene and the cell expresses the selectable marker; (2) incubating the cell under conditions whereby the cell or a progeny of the cell differentiates into a neuron comprising an axon or dendrites, and the neuron expresses the axon reporter under the transcriptional control of the gene; and (3) specifically detecting the axon reporter in the axon or dendrites.
In another embodiment, specific expression of the targeted gene product in the neuron is achieved using a binary system. This method comprises the steps of (1) transferring into the embryonic stem cell a gene trap vector comprising a polynucleotide comprising promoterless selectable marker and transcription factor encoding sequences under conditions whereby the polynucleotide integrates into the gene and the cell expresses the selectable marker and transcription factor under the transcriptional control of the gene; (2) transferring into the cell or a progeny of the cell a vector comprising a polynucleotide comprising a targeted gene product (such as an axon reporter) encoding sequence under the operative control of a transcriptional regulatory region (e.g. a promoter or operator sequence) activatable by the transcription factor; and (3) incubating the cell under conditions whereby the cell or a progeny of the cell differentiates into a neuron comprising an axon or dendrites, and the neuron expresses the targeted gene product under the transcriptional control of the gene, via the transcription factor. This system allows amplification of the axon reporter signal as well as creating a more versatile insertion which can be used to drive expression of any desired targeted gene under the operative control of the transcription factor.
Accordingly, the invention facilitates the identification of mutant phenotypes in the nervous system by including a cell-autonomous axonal marker, such as placental alkaline phosphatase (PLAP), in the vector, such that the projection patterns of neurons expressing the disrupted gene can be selectively visualized, in wild-type and mutant animals, by a simple histological stain such as for alkaline phosphatase activity (Fekete and Cepko, 1993; Fields-Berry, et al., 19
Leighton Philip A.
Mitchell Kevin
Skarnes William C.
Tessier-Lavigne Marc
Martin Jill D.
Osman Richard Aron
The Regents of the University of California
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