Specific aim #4:
To identify and characterize genes involved in axial extension, morphogenetic movements, axial patterning, and eye development via
A. chemical mutagenesis
B. insertional mutagenesis; and
C. To assess the feasibility of gene targeting in S. tropicalis by evaluating nuclear transfer from cultured somatic cells into enucleated eggs.
Rationale:
This proposal up to this point is already broad. While it is not unambitious to include mutagenesis, there are several reasons to conclude that the labor required for limited screens is outweighed by the likelihood of obtaining useful research tools and insights. First, mutagenisis by transgenesis (insertional mutagenesis) needs to be quantitated if only to optimize the transgenic procedure for generating stable lines, and offers a direct path to molecular characterization of mutant loci. Second, combining chemical mutagenesis with transgenesis- especially tissue-specific reporter lines- offers ways for us to focus our screens on the pathways that interest us, and may also make discerning subtly aberrant phenotypes easier. Third, the riskiest proposals, such as testing gene targeting in frogs, have very large potential payoffs relative to the investment of labor, since preliminary assays of feasibility can be conducted with available reagents (e.g. nuclear transfer from extant X. laevis euploid cell lines). Fourth, some of the same techniques that made early zebrafish screens feasible, including the ready production of gynogenetic haploids and diploids, likewise make it possible for us avoid carrying large numbers of animals for the full duration of the screen. Should the need arise, we have the facilities to maintain up to 20,000 S. tropicalis in a new facility recently constructed at the University of Virginia (see Grainger/Keller facilities).
The remaining projects in this proposal are feasibility studies, the results of which will determine subsequent investment of effort. While all of these projects have challenging goals, each is built around a preliminary experiment which does not require significant preparation, and which should reliably predict a project's long-term prospects.
Specific aim #4.B: Insertional Mutagenesis
The difficulty of cloning chemically-induced mutants is a drawback to most of the recently completed zebrafish screens. A small-scale zebrafish screen has avoided this difficulty by using a retroviral insertional mutagenesis approach32; virtually all of the mutants they have characterized have been rapidly cloned. In frogs, the ease with which large numbers of embryos transgenic at multiple loci can be generated suggests that insertional mutagenesis could be feasible.
We propose to assess the feasibility of gene traps as insertional mutagens. Gene traps are splice acceptor-containing constructs which, if inserted into an exon or intron, can simultaneously inactivate a locus and provide locus-specific reporter expression. This should enable us to screen F0 heterozygotes (the immediate products of a transgenic procedure using a gene trap vector) for candidate insertions in interesting pathways, greatly reducing the number of lines that we will carry to homozygosity for phenotypic analysis.
Large-scale gene-trap mutagenesis based on ES cells have been performed. One major effort involved screening ~40,000 integration-bearing ES colonies, approximately 1% of which expressed the gene-trap reporter and were assayed for expression pattern as chimeras, yielding 36 lines giving tissue-specific expression patterns at the developmental time point assayed 58. The frequency of transgenic frog embryos expressing gene trap reporters may be somewhat higher than 1%, since not all genes are expressed in ES cells, and since the frog transgenic procedure typically gives integrations at multiple loci1. Balanced against this is the evidence that gene trap insertions in ES cells seem to favor actively transcribed regions58, and it is not known how transgenic insertions are biased in sperm nuclei. Not all of the mutagenic insertions will give detectable reporter expression, however; integrations into exons, into genes with weak promoters, or in the wrong orientation or reading frame all could inactivate alleles without giving a reporter signal.
We believe that the frog system is amenable to this insertional mutagenesis strategy, but at greatly reduced expense relative to mice. We can generate multiple gene trap insertions in hundreds of transgenic animals per day for prompt reporter screening, discarding non-expressing tadpoles within a week. In addition, all stages of development in the frog are accessible and screening for marker gene expression can be done in living embryos at any stage.
Methods
Gene trap design:
All gene trap vectors contain a splice acceptor (SA) sequence followed by a marker gene. Since the marker gene lacks a promoter, it can only be transcribed and translated if it integrates properly into an exon or intron of an endogenous gene. The marker gene we have chosen is a very bright version of green fluorescent protein (GFP)90, which, unlike other reporter genes, is visible in living embryos. This allows us to detect expression at any stage of development without having to fix and stain the embryos; only embryos that express GFP in an interesting pattern need be cultured to maturity. In addition, since the trapped (and prospectively mutated) gene is always marked by GFP, one can easily distinguish between embryos carrying the mutation from ones that do not.
Preliminary experiments:
Since improving transgenesis is a major technical focus of the S. tropicalis genetics project, preliminary experiments to optimize efficient, multi-site integration in viable embryos are already underway. As with transgenesis using other vectors, it will be important to establish a balance between maximizing integrations and maintaining optimal long-term transgenic viability, since treatments that encourage incorporation of foreign DNA may also cause chromosome damage. Southern analysis to determine the absolute number of integration sites produced by each set of conditions provides an indirect assay for the prospective mutagenicity of gene traps under various conditions as an alternative to calculating the specific locus mutation rate. While maxima have yet to be determined, published data show 4-8 integrations per transgenic frog1. Extrapolating from the mouse ES gene trap screens, in which 1% of integrations led to expression of the reporter, we might expect ~4-8% of transgenic embryos to display locus-specific reporter expression. Thus a reasonable preliminary study might involve generation and fluorescence assay of ~1000-2000 transgenic embryos, or roughly one week of nuclear injections. The rate of production of embryos expressing GFP in specific tissues would determine whether injections would be scaled up.
Gene trap screen:
If preliminary experiments result in >0.5% embryos expressing GFP in a regulated fashion, ~10,000 transgenic embryos carrying the gene trap vector will be generated over the course of several months and screened for GFP fluorescence at stage 11, 17, 24, and 44. F0 transgenic embryos not showing detectable expression will be discarded; those displaying reporter expression, especially temporally- or spatially-regulated, will be categorized based on their reporter expression and sorted for culture to sexual maturity. Extrapolating a rough expected frequency of reporter expression from the mouse ES cell screens and transgenic integration rates, we might anticipate detecting 50-500 expressing lines. In order to determine whether an expressed gene trap has disrupted an essential gene, embryos will be cultured to sexual maturity and subjected to gynogenesis or androgenesis77 in order to score homozygous phenotypes, and outbred to carry the gene trap allele.
maternal effect genes:
Expressed insertions that do not display a recessive zygotic phenotype may have maternal effects124. Viable homozygous F1 gynogenetic diploid females will be raised to sexual maturity, and haploid, gynogenetic diploid, and outbred progeny will be assayed for mutant phenotypes.
Cloning trapped genes:
Identification of loci with insertions is straightforward. cDNA is prepared from tissues expressing the reporter and Rapid Amplification of cDNA Ends (RACE 118), using GFP sequence for the specific primer, is used to generate PCR fragments of GFP fusion transcripts. While there is a low probability of transcribed multiple insertions persisting after segregation, it should be possible to tell which are expressing the reporter by comparing GFP expression with whole mount in situ hybridizations using RACE probes. Multiple expressed traps would likely be reflected in pleiotropic and variable F2 haploid and gynogenetic diploid phenotypes; outcrossing for another generation and re-examing homozygous progeny should permit examination of single-gene phenotypes.
Potential pitfalls:
The main question is whether the gene trap will integrate and express the GFP reporter at a sufficiently high frequency to be useful as a mutagen. One adaptation to mammalian gene traps, the use of a viral Internal Ribosome Entry Site (IRES)125 upstream of the reporter to provide expression in all three reading frames, may not function in amphibians (D. Turner, pers. comm.). Enhancer trap constructs containing minimal promoter elements are less stringent in their requirements for tissue-specific reporter expression, but are less likely to be mutagenic. The pseudotyped retroviruses that were used for insertional mutagenesis of zebrafish also infect cultured frog cells32, 126 and may be an alternative reagent for insertional mutagenesis.
timeframe:
construct gene trap vector 11/97; optimize insertional efficiency 2/98-4/98; production and screening of F0 transgenic females 6/98; phenotype analysis of F2 haploid and gynogenetic diploid progeny 11/98
implementation:
laboratory of Enrique Amaya