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.C Gene Targeting
Rationale:
A large number of candidate genes for participation in developmental processes have already been identified in Xenopus and other systems. Gene function in many of these processes could be effectively characterized in advanced frog embryological assays if specific mutants were available. While candidate gene mutations may be characterized through the insertional and chemical mutagenesis screens described in specific aims #4A and B, the efficiency with which a mutant for any given candidate is identified is likely to be very low. Currently, mouse embryonic stem cell (ES cell) technology offers the only system for targeted mutagenesis in higher eukaryotes.
4.C.i. Nuclear transfer from cultured cells:
ES cell equivalents are unlikely to be reproduced in amphibian systems soon. However, late-stage embryos and in some cases fertile adult frogs can be produced by nuclear transfer from a surprisingly wide variety of donor cells, including endoderm, melanophores, keratinized skin cells, neural cells, erythrocytes, and intestinal epithelial cells127-129. If a cultured cell system could likewise be developed as a source of nuclei, then identifying and expanding cells that carry specific mutations and using them to make transgenic mutants may become feasible. What are the requirements for developing homologous recombination using nuclear transfer?
1. A cell line whose nuclei are competent for transfer into enucleated eggs and direction of subsequent development;
2. an efficient transfection system; and
3. a method for identifying and cloning cells which have correctly integrated targeting constructs. Selection regimes simplify this last task, but are not formally required.
Of these requirements, the first is likely to be the most stringent. Several cell lines have been assayed in serial nuclear transfer protocols without producing feeding-stage tadpoles130, 128,131. The high frequency of aneuploidy in long-term cultured cells130 has been a significant stumbling block; in only one of these cases, using a haploid cell line, were the cells identifiably euploid131. Developmental potential of ES cells likewise becomes restricted with multiple passages28; by focusing our manipulations on low-passage cultures, karyotypic abnormalities may be minimized. Encouragingly, we have recently generated and characterized two clonal X. laevis cell lines, one with fibroblast-like morphology, one with epithelial morphology, from dorsal explants of neurulae. Both of these lines are karyotyically euploid with respect to chromosome number (>60% of mitotic nuclei displaying thirty-six chromosomes) (see Preliminary Results). Even when euploid somatic nuclei are transplanted, visible chromosome aberrations appear in a large fraction of the resulting embryos127, perhaps as a result of the rapid post-fertilization cell cycle, and are thought to be a principal impediment to normal development. Strategies that may help protect nuclei from these stresses include serial transplantation128, incubation in meiotic oocytes or activated egg extracts1, and induction of quiescence by serum starvation129. The latter was implicated in the recent success in cloning by nuclear transfer from a sheep cultured cell line, in which it was hypothesized that the chromatin of quiescent nuclei was more readily modified by the oocyte cytoplasm.
We propose to test the prospects of gene targeting in S. tropicalis by generating and karyotyping cell lines and assaying their developmental potential in a nuclear transfer procedure adapted from the transgenic technique. If successful, we will elaborate an effective electroporation procedure, and test selection protocols and methods to identify homologous recombinants in vitro and in transgenic frogs.
Methods
Preliminary experiments:
Two clonal cell lines have already been established from X. laevis neurulae and shown to have a euploid complement of chromosomes (see Preliminary Results). A modified transgenic protocol will be used to assay nuclei from these lines for their ability to direct development of eggs that have been UV-irradiated to inactivate the maternal genome134. Methods for nuclear transfer from cell lines have been streamlined130; variations such as serum starvation and mitotic extract incubation may be evaluated with relatively little effort. Identification of a developmentally-competent source of X. laevis cultured cell nuclei would encourage us to establish comparable euploid S. tropicalis lines, and to develop transfection, targeting strategy, and selection protocols with which to isolate clones bearing targeted alleles. Two positive selective regimes, neomycin resistance and hygromycin resistance, have already been worked out in Xenopus cell lines126; 130. Additional cell lines may be established from isogenic S. tropicalis neurulae using established methods79, 135, 136 as described in Preliminary Results. Further experiments with cell lines will be predicated on the identification of euploid cell lines with developmentally-competent nuclei.
potential pitfalls:
If euploid developmentally-competent nuclei from established cell lines turn out to be unobtainable, it may be possible to perform gene transfer and selection in low-passage cultures of primary cells. Given the ease with which large numbers of transgenic embryos can be constructed by transfer of sperm nuclei, it may be feasible to use donor nuclei from cultured cell populations that are enriched, but not clonal, for the correct integration, and then sort through transgenic products to identify targeted embryos. Preliminary experiments for this alternative include evaluating nuclear transfer protocols from mixed primary cultures of neurulae.
timeframe:
assay X. laevis cell lines by nuclear transfer 10/97; characterize S. tropicalis cell lines 8/98
implementation:
De Simone lab, generation and characterization of cell lines; Grainger lab, nuclear transfer