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Progress Report: Oct. 2005

As part of the NIH RFA for X. tropicalis genetics, we have periodic meetings to discuss our progress and the progress of other labs developing X. tropicalis genetics. Our latest progress report is below. Previous progress reports are also available.

Date
Most Recent Oct 2005
Apr 2005
Sept 2004
Apr 2004
Oct 2003

Progress report for October 2005

 

Summary:

1. Mutagenesis protocol(s) that you are using and the outcomes that they

produce

Our primary mutagenesis has been with gamma-irradiation of sperm suspensions. We are now pursuing ENU mutagenesis of spermatogonia in addition.

Gamma ray mutagenesis. We have approximately 1000 g-ray mutagenized F1 founders that are now becoming sexually mature. We used a dose of 600 rads to mutagenize sperm. This dose results in a maximal mutation rate (1% hit rate) and still preserves fertility (http://tropicalis.berkeley.edu/home/mission/RMH-Mutagenesis.html). We are now generating our F2 families. In the F1 founders, we identified over 30 adult frogs that had developmental defects and may be harboring dominant mutations. We are testing the genetics now. Currently, our screening efforts have confirmed 14 recessive mutations and identified another 4 potential recessive mutations. Descriptions of these mutations are on our website (http://tropicalis.berkeley.edu/home/genetic_resources/mutants/mutants.html).

 

ENU mutagenesis.We have initiated a pilot ENU mutagenesis screen. Our previous pilot screens were complicated by the prevalence of background mutations in the original frogs; however, for this pilot screen, we are using a frog line (Golden) that has so far been shown to be free from background mutations. We mutagenized spermatogonia by injecting males with 100mg/kg of ENU between one to three times weekly. We are now testing each of these three doses to identify which is optimal for survival, fertility, and mutation load.

Once the optimal dose is determined, we will initiate an ENU mutagenesis screen in a similar fashion as the g-ray mutagenesis. Animals generated from the sperm mutagenesis will also be prepared for reverse genetic screening by sequencing defined loci to identify mutations and also quantify hit rates.

 

2. Screening strategies that you are using and the phenotypes that you've

identified

Our screening strategy is the same as outlined in the last progress report. Briefly, we are doing a standard F3 screen. We will be screening six F2 females and six F2 males by backcross matings only. Embryos from screen matings are raised through to swimming tadpoles (4 days postfertilization) and any phenotypes scored by morphology. Our strategy is outlined in prior reports and on our website (http://tropicalis.berkeley.edu/home/mission/phenotyping.html). Because sperm mutagenesis can produce mosaic founders, we will pursue any phenotypes that are seen in at least 10% of the embryos. For founders whose spermatogonia were mutagenized with ENU, mosaicism will no longer be an issue allowing for both backcross and sibling matings in the F2s. Phenotypes should be present in typical Mendelian ratios.

We have identified mutations in cardiovascular/lymphatic development, craniofacial development, melanocytes/cell migration, eye development, germ layer differentiation, early viability, cell proliferation, and skin pigmentation.

 

3. A list and description of the mutants that you've identified

confirmed mutation - mutation has been propagated at least one generation past the founders.

potential mutation – mutation has been seen in the founders at least once. Typically, the mating is repeated to see if the phenotype persists and then outcrossed to see if the phenotype can be recovered in the next generation. If it can, then the mutation becomes confirmed.

background mutation means that we identified the mutation in individuals that we have not exposed to any form of mutagenesis.

 

Mutations on the Nigerian line:

grinch: confirmed background mutation in lymphatic development

curly: confirmed background mutation in cell proliferation/survival

bubblehead: confirmed background mutation in craniofacial development

bub2: confirmed background mutation in craniofacial development

loris: confirmed background mutation in craniofacial and eye development

jawbreaker: confirmed background mutation in craniofacial development

beaver: confirmed background mutation in embryo viability

rubenesque: confirmed mutation in pigmentation, cardiovascular function and cell migration

hourglass: confirmed mutation in eye development

owl: confirmed mutation in craniofacial development

blind ambition: potential mutation in dorsoventral development

juanito: potential mutation in melanocyte and gut development

dexter: potential background mutation in left:right patterning

clearview: potential mutation in melanocyte development

undulated: potential mutation in dorsoventral development

 

All of the mutants above are recessive. In addition to these we are determining whether defects seen in our latest round of F1 mutagenized adults are dominant. Currently we are pursuing about 40 individuals that have a variety of defects in skeletal/limb formation, pigmentation, eyes, and locomotion.

 

Mutations on the Ivory Coast line:

Ivory Coast (Virginia)

curly: confirmed background mutation in dorsal/ventral axis formation. This has been identified in an individual from the Ivory Coast line developed from Virginia stock 239 and was shown by complementation testing to be allelic to the Nigerian curly mutation.

 

Ivory Coast (TGA)

bubbletrouble: confirmed background mutation in craniofacial development. This was found through inbreeding a TGA Ivory Coast pair derived from the colony in France.

axanthic: confirmed pigmentation mutant believed to be lacking xanthophore pigmentation. This was found through inbreeding of the TGA line.

return-of-the-redeye: potential mutation in cardiovascular development found through inbreeding a TGA Ivory Coast pair derived from the colony in France.

 

 

4. an estimate of the mutagenesis rate

Results from testing 600 rads g-ray mutagenesis suggest a single locus is mutated in approximately 1 in 100 embryos that survive past gastrulation and produces fertile F1 animals. The details of this were outlined in the previous report (April 2005).

         The mutagenesis rates for ENU spermatogonia mutagenesis are being determined now using the same strategy as for gamma mutagenesis (explained in detail below in this report). Preliminary results suggest that 3 doses of ENU (the highest we gave) gives mutagenesis rates similar to our optimized gamma ray dose. As may be expected, the third dose significantly increases the level of mutagen load over 2 doses. Clearly 3 doses so far is optimal since the mutagenized frogs are fertile and have the highest mutation load, but we still need to determine if additional mutagen would be optimal.

 

5. a description of how people can get your mutant strains

We are ready to distribute curly and grinch mutants. We will soon be able to distribute bubblehead, jawbreaker, beaver, and hourglass. We have sent grinch frozen sperm to the Zimmerman lab for propagation in England.

In addition, we are ready to distribute non-mutant inbred animals from five different frog lines: Nigerian, Golden, Ivory Coast (Virginia), Ivory Coast (TGA from France), and Population A (http://tropicalis.berkeley.edu/home/genetic_resources/Inbred-strains/inbred-strains.html). Requests should be made to harland@socrates.berkeley.edu.

We have sent Golden frogs to Japan for further inbreeding there. We sent Golden, TGA, and PopA frogs to the EPA in Minnesota and sent Nigerian and TGA frogs to Dr. Zorn at the Cincinnati Children’s Hospital. We are now preparing to ship Golden strain frogs to Dr. Pollet’s group in France for further inbreeding there.

 

6) time required to get animals to sexual maturity, and procedure used to

achieve the shortest time.

Females take longer than males to mature: females in 5-7 months; males in 3-4 months. For our husbandry methods, see http://tropicalis.berkeley.edu/home/. We have ongoing selective breeding programs for all of our strains in order to promote fast sexual development.

 

7) Publications and manuscripts supported in part by this grant since the last update (April 2005)

U. Hellsten, M. Khokha, T.C. Grammer, R.M. Harland, and D. Rokhsar  Subfunctionalization of Pairs of Xenopus Co-orthologs Following Whole Genome Duplication in Xenopus laevis (in preparation)

 

DD Roche, JF Cheng and RM Harland. Characterization of alpha globin genes in Xenopus tropicalis. (in preparation)

 

Maresca, T., Brown, K., Grammer, T. and Heald, R. Mitotic extracts from Xenopus laevis and X. tropicalis show dynamic determination of spindle length (in preparation)

 

Fletcher, R. B., Baker, J. B., and Harland, R. M. FGF8 spliceform-specific activities: FGF8 spliceforms mediate early mesoderm and posterior neural tissue formation in Xenopus Development (submitted)

 

Wills, AE, Harland, RM, and Khokha, MK. Twisted gastrulation is required for forebrain specification and cooperates with Chordin to inhibit BMP signaling during X. tropicalis gastrulation.  Dev. Biol. (submitted)

 

Choi, VM, Harland RM, and Khokha, MK.  Developmental Expression of FoxJ1.2, FoxJ2, and FoxQ1 in Xenopus tropicalis. Gene Expr. Patterns (submitted)

 

A Mve-Obiang, RE Lee, ES Umstot, KA Trott, TC Grammer, JM Parker, B Ranger, R Grainger, EA Mahrous and PLC Small. A newly discovered mycobacterial pathogen isolated from lethal infections in laboratory colonies of Xenopus species produces a novel form of the M. ulcerans macrolide toxin, mycolactone. Infection and Immunity (2005) 73(6), p3307-3312


X. tropicalis genetics and genomics

Progress report for October 2005

 

Timothy C. Grammer, Mustafa K. Khokha, and Richard M. Harland

Department of Molecular and Cell Biology

University of California-Berkeley

401 Barker Hall

Berkeley CA 94720

 

Gamma-Ray Mutagenesis

Optimized g-irradiation mutagenesis screen ongoing

We used the Golden line as the starting point for our large g-ray mutagenesis screen. To date, no background mutations have been found in the Golden line, which has now been inbred for four generations. A month prior to sacrificing the males for mutagenesis of sperm, we mated them to four females to ensure that the males were fertile and did not carry background mutations that would interfere with our large screen. Using these four independent founding mothers, we fertilized eggs with sperm that had been exposed to 600 rads g-irradiation (using our previously reported protocols).

Following two sessions of mutagenesis and in vitro fertilization, we have raised over 1,000 F1 frogs. The first set of F1s is now 10 months old (>100 frogs) and the second set (>900 frogs) is now 7.5 months old. We are now generating F2s and will screen them by backcross matings to their F1 parents. For details on how we plan to screen, please refer to our previous progress report (April 2005).

We surveyed our F1 frogs for morphological or behavioral defects. We identified over 40 individuals that had a range of defects including: deformed skeletons, eye dysmorphology, limb abnormalities (extra limbs, missing limbs, deformed limbs, digit defects), pigmentation defects, and abnormal swimming behavior. Frogs raised at the same time from unirradiated sperm showed no such defects. The defects were frequently only apparent unilaterally, which may indicate mosaicism. We are now generating F2 families from these individuals to determine if they are carrying dominant mutations that should be apparent in the F2 froglets.

ENU mutagenesis

Sperm mutagenesis

We did three trials of ENU mutagenesis using homozygous gamma-crystallin GFP males that are free from background mutations. Sperm suspensions were mutagenized with 0, 0.2, 2, and 10 micromolar doses of ENU for 10 minutes at room temperature using a protocol adapted from Lyle’s and Rob’s labs. We then used this sperm to fertilize the eggs of double heterozygous female carriers of the recessive mutations grinch and curly. In this way, we wanted to score for the mutations of three separate loci (GFP, grinch, and curly) to titer the mutation rate at these ENU doses.

            Unfortunately, the ENU treatment caused a high rate of gastrulation defects and although many embryos were obtained, we could not quantify the mutation rate due to the prevalence of general developmental defects. At the low doses, tadpoles were obtained, but no mutants were scored. We are contemplating future attempts to overcome these setbacks, but may simply move to spermatogonial mutagenesis, which has been more promising.

Spermatogonial mutagenesis

ENU spermatogonial mutagenesis screen used Golden strain frogs that have so far been shown to be free from background mutations. We mutagenized the spermatogonia of these males via dorsal lymph sac injections of ENU in a protocol adapted from Lyle’s, Derek’s, and Rob’s labs. A dose of mutagen of 100mg/kg was given once (1x), twice (2x), or three (3x) times at weekly intervals. We therefore have four groups of sibling males: 0 ENU, 1xENU, 2xENU, and 3xENU.

            After resting the frogs for at least a month after the last ENU mutagenesis, we mated the males every month to “clear” the mutated sperm. We found that all the mutagenized males went through one to two initial rounds of fertilization resulting in deformed embryos followed by two to three rounds of failed fertilizations. We expected the infertility was due to the “clearing” of heavily mutated mature sperm. The males, at all three doses used, have now regained their fertility and we predict are carrying mutated sperm non-mosaically that have arisen from spermatogonial mutagenesis.

            We are now testing the mutagenesis rates of the ENU using a protocol similar to the one we used for the gamma-irradiation and the ENU sperm mutagenesis analysis. Briefly, we mated the males to double heterozygous female carriers of the recessive mutations grinch and curly and assayed the number of grinch and curly embryos.

            We haven’t tested all of our males, but the preliminary results are promising. We find that all males tested so far are fertile. Our findings suggest that the highest dose of mutagen used (3x) is the best tested to date (see table). Our 1x mating showed no significant mutagenesis of the curly/grinch loci, 2x showed slight to no significant mutagenesis of the curly/grinch loci, but 3x showed a significant increase (to an apparent 1% hit rate) of mutagenesis at the curly/grinch loci. We were very stringent in our “calling” of the curly and grinch phenotypes so we are confident that if anything, we are underestimating the possible mutagenesis rates. These preliminary scores will be improved by further test crosses.

 

ENU dose             

curly hit rate (n)

grinch

Total counted

0

0.2% (1)

0%

813

0

0%

0%

2188

0

0%

0.1% (1)

1962

1x

0%

0%

217

2x

0.3% (3)

0.4% (4)

2127

2x

0.3% (3)

0%

2155

3x

1% (2)

0%

400

3x

0%

1% (2)

391

 

From our results so far, we anticipate that a dose of at least three injections of 100mg/kg per injection will be best and we may try higher mutagen doses. Once the optimal dose is determined, we will initiate a large ENU mutagenesis screen of spermatogonia that will be performed in a similar fashion as to our strategy being used for the gamma-ray mutagenesis. Animals generated from the sperm mutagenesis will also be prepared for re-sequencing as part of a reverse genetic strategy to identify mutated loci.

 

Mutants

Please see our previous report (April 2005) for descriptions of the mutants grinch, curly, bubblehead, loris (previously called bub9(bigeye), jawbreaker (previously called agnatha), beaver, hourglass, owl, clearview, dexter, and bubbletrouble. For pictures of mutants, please see our website at:

http://tropicalis.berkeley.edu/home/genetic_resources/mutants/mutants.html.
In general, while we keep some adult animals on the Nigerian background, we have been outcrossing the propagated animals to Ivory Coast strains so that they will be useful for mapping.

 

Recent analysis of mutants

grinch: We believe that the grinch mutation may be a defect in the development of the lymphatic system, possibly of the lymph heart. We have found that the heart, blood, and cardiovasculature appear to develop normally and we know that the edema is not due to the pooling of blood, but rather is clear fluid that accumulates pericardially and around the pronephric region in the area of the lymph heart. We are now focusing on the development of the lymphatic system for this mutant.

Meanwhile, we have mature grinch mapcross animals that are a hybrid between the Nigerian strain (carrying the mutation) and the Ivory Coast TGA strain. We identified which of these F1 mapcross adults are carriers of grinch. We mated them and collected grinch and wildtype embryos that we have isolated DNA from and are now ready to map the mutation once genetic markers become available.

 

curly: We believe that the curly locus may encode an antagonist of cell proliferation. We find that curly mutants suffer from a failure of cells to differentiate and see a dramatic increase in both phospho-Histone3 positive(proliferating) cells and concurrent apoptosis in all tissues. We are now focusing on this defect.

We also have mature curly mapcross animals and have collected DNA from curly embryos for mapping.

 

loris: We showed by complementation testing that bub8 and bub9(bigeye) are allelic. This mutation is now being called loris to differentiate it from our other craniofacial mutants we have been calling “bubs” due to similarities to our first craniofacial mutant bubblehead. We have shown that loris is nonallelic with bubblehead, bub2, and bub5(dexter). The loris mutant embryos are now being characterized.

We have mapcrossed this mutation and are now raising these animals.

 

dexter: We showed by complementation testing that bub5(dexter) is nonallelic to the other bubs and we are now simply calling this mutation dexter. We only have a few individuals of this mutation and are producing offspring to generate more carriers as well to produce mutant embryos for analysis.

 

jawbreaker: The jawbreaker mutant was previously known as agnatha. After analyzing the cartilage of jawbreaker, we now know that all of the head cartilage structures do form, but that meckel’s cartilage and the ceratohyal cartilages are greatly deformed. We have analyzed numerous markers of early neural crest development and all are normal. We currently believe that this mutation is in the regulation of growth of the cartilaginous regions of the head and is not a defect of neural crest induction or migration. Since the mutants do not lack, but have defective jaw structures, we have renamed the mutant jawbreaker.

            We have mapcrossed this mutation and are now raising these animals.

 

NASA Ames frogs: juanito, undulated, and blind ambition

Dr. Sigrid Reinsch at NASA Ames Research Center has been performing an enhancer trap screen with the GFP construct made by Enrique’s group. In mating a clutch of frogs from transgenic founders, her group noted that one family of frogs occasionally produced tadpoles that lacked pigmentation. They were unable to raise these frogs, but wondered if they might be albinos. They did note that the pigmentation defect was not due to a GFP insertion since it did not cosegragate with any GFP fluorescence.

            Due to the importance of finding an X. tropicalis albino strain, we obtained these frogs from her lab and mated them. We did find one pair that produced the phenotype seen in her lab, but it is not a simple albino. The mutation, called juanito, is an embryonic lethal mutation causing a lack of pigmentation (both retinal and melanocyte), gut coiling defects, and blood pooling. The embryos die by stage 46. We have confirmed in three matings that the phenotype is reproducible, but is not seen in Mendelian percentages but rather is consistently around 15% of the embryos. We are now mapcrossing the female we believe is carrying the mutation.

            In setting up sibling matings looking for the juanito phenotype, we believe we have uncovered two other mutations. One mutation, undulated, is seen in the same pair that is carrying the juanito phenotype. The undulated mutants have severe axial kinking (similar but distinct from curly) and die around mid40s. The repeated presence of undulated in the matings of the parents and the percentages of wildtype (55%) and juanito (15%) embryos in these matings suggests that the parents may be double heterozygous for two mutations: juanito and undulated. As stated for juanito, we are mapcrossing the female and will follow the inheritance of these phenotypes in the next generation.

            Also, while setting up matings for identifying juanito, we found two sibling pairs that did not have juanito or undulated, but did possess a different mutation we are calling blind ambition. This phenotype suffers from axial kinking and deformation of posterior dorsal structures (tail, somites, notochord, neural tube). Interestingly, all mutants were GFP positive in locations where embryonic defects were present. Some wildtype embryos were also positive for GFP but these embryos appeared to have a lower intensity of the GFP compared to mutants, suggesting that they may be heterozygotes. Therefore, although this is very preliminary, we believe that this may be an insertional mutation causing a recessive trait. We are pursuing this now and will try to clone the mutation by either inverse pcr or 5’ RACE. It is interesting to note that while many have abandoned transgenic gene trap strategies for mutagenesis that in this one line we have identified a possible gene trap mutation as well as two other mutations.

 

Ivory Coast TGA mutants: bubbletrouble, return-of-the-red eye, and axanthic.

            During our inbreeding of the Ivory Coast TGA line (obtained from Dr. Pollet’s group in France) we have found two confirmed and one potential mutation.

bubbletrouble: This is a confirmed background recessive embryonic lethal mutation in craniofacial development. We identified this mutation by sibmatings in a TGA Ivory Coast pair. The embryos suffer from blisters and edema that originates in multiple places simultaneously. The bubbletrouble embryos die by mid40s.

            We are mapcrossing the carriers to our mutant-free Nigerian strain for further propagation, analysis, and mapping.

return-of-the-redeye: This is a potential background recessive mutation in cardiovascular development found through inbreeding a TGA Ivory Coast pair derived from the colony in France. The embryos suffer from cardiovascular defects resulting in large amounts of blood pooling in various tissues. We are remating the pair of frogs that showed this phenotype to see if it repeats. If it does, we will mapcross them to the Nigerian strain for further work.

axanthic: This is a confirmed viable recessive pigmentation mutant believed to be lacking xanthophore pigmentation, giving the adults a bluish cast. This was found through inbreeding efforts of the TGA line. The phenotype is an adult phenotype but is difficult to distinguish prior to metamorphosis. Our frog facility supervisor Maura Lane found the phenotype, and she is characterizing and breeding the frogs now. Maura noted that a pair of TGA frogs (which were green in color) was consistently generating bluish-gray offspring at a rate of about 17-19% albeit in small clutches.

            In subsequent matings, blue-blue matings always generate 100% blue colored frogs. Green-green matings have been found to generate either 100% green frogs, or 25% blue frogs. In our only green-blue mating so far, all offspring were green. We therefore believe that the blue coloration is a single locus trait that is recessive to the green coloration. Obviously, our data suggests that our green frog population consists of green/green homozygotes and green/blue heterozygotes. Maura is now testing our colony to identify the genotypes of the frogs.

            Our current hypothesis is that this phenotype is caused by axanthism (a lack of xanthophore pigmentation) either due to a loss of xanthophores or a defect in the production of pteridines, the pigmentation of xanthophores. There is a great deal of literature on axanthic salamanders and fish due to a variety of autosomal recessive mutations. We are pursuing this analysis now. We are mapcrossing the axanthic frogs to the Nigerian strain for future positional cloning.

 

 

Inbreeding plans

We are continuing to inbreed our frog strains. Currently, we have generated F10 Nigerian, F8 Ivory Coast (Virginia), F4 Golden, F3 Population A, and F3 Ivory Coast (TGA).

We are selectively breeding the individual males that most rapidly develop nuptial pads. We start ovulating the females around 5 months of age and selectively mate those females that most quickly generate good quality eggs.

In order to generate inbred lines at a faster pace, we are now doing gynogenesis on the Ivory Coast (TGA) and Population A lines. We are using the late cold shock procedure given to us by Rob’s lab. This technique would normally generate only males. However, to produce females, we are raising the gynogenic tadpoles in estradiol according to Lyle’s recommendations. We are now raising these females (that will be genotypically male). Currently we have about five tadpoles from the Population A line growing up that look quite healthy. In the next few months, we will perform another round of gynogenesis with both Population A and Ivory Coast TGA to raise additional tadpoles.

Once they are mature, we will do a second round of gynogenesis using early cold shock and will raise half of these offspring normally and the other half in estradiol. From each female, we will then produce a clutch of isogenic animals: males and sex-reversed females that can be used to perpetuate the line. We will then confirm isogenicity by AFLP analysis.

 

Transgenics
We have been trying out various potentially simple methods for generating transgenic animals, since an easy transgenic procedure will be a valuable tool.
Meganuclease

This system was published to work well in Medaka. Several zebrafish labs told us that they were adapting it for their system. In addition, we have been encouraged by other Xenopus labs that are getting it to work.

            We first started with a self-made vector we obtained from a nearby zebrafish lab. They constructed a transgenic vector by inserting I-Sce1 meganuclease sites flanking the multiple cloning site of the Bluescript vector. We inserted the Pax6-GFP and Otx2-GFP promoter-reporter genes that had been shown by Lyle to work in sperm nuclear transfer experiments. We then followed a zebrafish protocol given to us that was based on the published Medaka protocol.

Our results showed a high number of fluorescent embryos when simply injecting the DNA alone. This number increased when the DNA was coinjected with the I-Sce1 enzyme (Roche). We saw the same effects in both Xenopus laevis and Xenopus tropicalis. The fluorescence was predominantly localized to the somites, neural tube and eyes. The area of fluorescence was not uniform, but often was a salt and pepper pattern with some individual cells being very bright.

All of these embryos lost fluorescence upon raising them to one week of age, so we believe it was episomal expression (from non-integrated DNA). We therefore set out to improve the procedure for Xenopus. We used midiprepped and maxiprepped DNA since we had heard from Paul Meade’s lab that miniprep DNA does not work well for SB transgenics. We phenol:chloroform extracted the DNA as recommended by Rob’s group. We then did an extensive panel of titration experiments with differing ratios of DNA to enzyme and at various injection concentrations and volumes. All of these resulted in mostly what we believe to be unstable, episomal expression. Just in case, we are raising some of the Xenopus tropicalis embryos that showed the brightest fluorescence. They are now frogs and will be mated as soon as they are mature to determine if any of the transgenes have been propagated.

Meanwhile, we have been in contact with Rob and Hajime from Rob’s lab. We noted that we were using some different procedures, which we then changed to reflect what Rob’s lab has done. Most notably, we obtained a new meganuclease vector from Rob’s lab and subcloned the Pax6-GFP and Otx2-GFP genes into this vector. In addition, we obtained a zebrafish alpha-tubulin GFP construct already in a meganuclease vector originally coming from the Medaka group that published the procedure. Unfortunately, we have not been able to get this system to work and hope to discuss these issues further at the October meeting.

Tol2

We are also testing the Tol2 transposase system using the protocols from Paul’s lab. We have only just begun these experiments using the EF1-alpha GFP construct that was in the published Xenopus Tol2 paper. In general, we see lots of mosaic expression. Many of the injected embryos die or have developmental defects, but of the healthy ones, generally a high proportion are fluorescent (as high as 70%). We use 1 ul Tol2 transposase RNA (1 ng/ul) in a 10 ul reaction containing 1 ug of EF1-alpha GFP DNA. We are injecting about 3

nl/embryo. Lower concentrations lead to fewer fluorescent embryos and

faint expression. We still see salt-and-pepper episomal expression. Injections with DNA alone have fewer fluorescent embryos and the fluorescence is generally not as bright and is more scattered than when DNA is co-injected with the transposase RNA. We are now raising the tadpoles in order to test germline transmission.

 

Other work

This grant has also been important in stimulating our related work with Xenopus tropicalis. Most of these experiments are supported by other grants, but have benefited from the Xenopus tropicalis colony that was established by this grant. However, two of these projects are worthy of note, since our contribution has been supported by this grant. The first has exploited sequence comparisons of Xenopus tropicalis and Xenopus laevis and is a collaboration with Dan Rokhsar’s group. They examined X. laevis genes that show expression of both pseudoalleles, and found several which show differences in expression, based on EST abundance. We have taken some of these and examined expression patterns, using diverged 3’UTR sequences. Out of 14 tested, four showed different spatial expression patterns. Comparison of genomic sequences may lead to identification of the determinants of these different patterns.

In a second collaboration, we have been exploiting Xenopus tropicalis egg extracts. We were initially prompted to do this by the hostility of some in the Xenopus laevis cell biological community to the sequencing of X. tropicalis. Our view was that it was simpler to adapt X. tropicalis to cell biology than it would be to generate comprehensive sequence information for X. laevis. We helped Rebecca Heald’s lab to make extracts, and they have exploited these to make the novel finding that the size of the mitotic spindle is determined dynamically. Previously it was thought that spindle size was determined by a fairly rigid scaffold, at least until anaphase, but mixing of X.tropicalis and X. laevis extracts (which determine different spindle sizes) showed that spindle size changes in less than a minute upon mixing a trop extract with that of laevis (or vice versa). Heald’s group has also done some preliminary mass spec analysis of trop proteins, and finds a much cleaner spectrum of peaks from the trop extract, consistent with the diploidy. We expect that X. tropicalis extracts will be very useful for proteomic analysis, given the genome sequence predictions of a proteome, and this simpler spectrum of protein sequences than is found for X. laevis.

Website Updates

 

Updated sections:

Husbandry updates including analysis of Marine Biotech system

Immunocytochemistry updates

Mutant updates

Sperm freezing update

 

New sections:

Histology section

Internal search engine

SSLP mapping protocols

Apoptosis/TUNEL section