WO2002081680A1 - Method for producing transgenic teleost fish - Google Patents

Abstract

The invention relates to a method for producing transgenic teleost fish by intracytoplasmic injection of eggs with non-decondensed sperm nuclei which have been pre-incubated with the transgene DNA. Integration of the transgene into the germ line and non-mosaic expression of the transgene is achieved using this method.

Description

METHOD FOR PRODUCING TRANSGENIC TELEOST FISH

BACKGROUND OF THE INVENTION

1. Technical Field

This invention relates to the field of molecular biology, and specifically to transgenic animals and methods for their production. In particular, a method is provided for producing transgenic teleost fishes, such as zebrafish.

2. Description of the Background Art

Teleost fish, and in particular, zebrafish, are becoming a widely used organism for studies on vertebrate development as well as a model system for disease, primarily because of the ease of obtaining mutations which affect diverse processes. Some degree of genome manipulation also has been possible: foreign genes have been introduced into the teleost germline using a variety of methods such as injection of DNA into the early embryo (1), transposon-mediated gene insertion (2), or retrovirus-mediated gene transfer (3) . These methods have all been shown to give rise to stably integrated transgenes, but the rate of transmission is low. Integration of foreign DNA usually occurs late using these methods, so that expression is mosaic, and only a small proportion of embryos contain the transgene integrated into the germ line.

One approach that circumvents the problem of mosaicism as well as low transmission rates is fertilization of eggs by injection of sperm that carry a transgene. This approach has been used successfully in Xenopus laevis (4) and Xenopus tropicalis (5) . These methods involve restriction enzyme- mediated integration of the transgene into sperm nuclei which have been decondensed by incubation in egg extract. Large numbers of non-mosaic transgene-cxprβssing individuals can be produced in this way. Perry et al. (6) recently reported that transgenic mice can also be produced by intracytoplasmic sperm injection.

Telεosts such as zebrafish pose several technical difficulties for the injection of sperm nuclei: the egg is protected by a tough chorion, and the sperm nucleus needs to be placed near the animal pole, where the female pronucleus is localized. Injections thus cannot be as rapid as with Xenopυs eggs, which have a more obvious polarity. Also, smaller needle bores are required, since some teleost eggs, such as the ^■ zebrafish eggs, are considerably smaller than that of Xenopus laevis . The methods developed for Xenopus transgenics have not been successfully used in zebrafish, in part due to the large size of the needles which must be used when injecting the larger decondensed sperm nuclei.

Fertilization by intracytoplasmic sperm injection has been used in other species _r including rhesus monkey (8) and mice (6) . Perry et al. have reported an intracytoplasmic sperm injection approach to transgenesis of mice (6). These methods involved a one minute incubation of the sperm nuclei with the exogenous DNA and injection with a piezzo-injection apparatus which was used to pick up and inject individual sperm nuclei one at a time. This method was found not to be effective with zebrafish, possibly because of differences in early development. Early cleavages occur much more rapidly in teleosts than in mice, leaving less time for DNA integration to occur.

Methods which have been successful in Xenopus have used decondensed sperm for injection, which requires a large bore needle. This method is not useful for zebrafish. Large bore injection needles result in leakage of cytoplasm from the egg after injection, usually causing abnormal development.

The creation of transgenic lines using prior art methods such as DNA injection result in incorporation of the transgene in the germ line in only about 5 to 10% of the injected animals. Thus, only a small proportion of FI individuals are transgenic. Experiments requiring transgenic embryos can only be performed with offspring of F2 fish using these methods. This requires approximately 6 to S months for production of the embryos in teleosts such as zebrafish. A method of trangenesis which gives rise to fish with the transgene integrated in all cells, would allow the offspring of the F0 fish to be used for experiments, saving 3 to 4 months.

A method, for reliably producing transgenic teleost fish having non-mosaic expression of the transgene is lacking in the art.

SUMMARY OF THE INVENTION Accordingly, this invention provides a method of producing a fertile transgenic teleost which comprises providing a suspension of demembranated sperm nuclei wherein the sperm nuclei are not decondensed and a DNA transgene, incubating the DNA transgene with the sperm nuclei under conditions such that the DNA transgene is taken up by the sperm nuclei, providing mature eggs of the same species as the sperm nuclei, injecting the incubated sperm nuclei into the mature eggs using an injection needle having an outside diameter of about 10 μm to about 15 μm, and discarding injected eggs which exhibit no cleavage or abnormal cleavage. In another embodiment, the invention provides a method of producing a fertile transgenic zebrafish which comprises providing a suspension of demembranated zebrafish sperm nuclei wherein the sperm nuclei are not decondensed and a DNA transgene, incubating the DNA transgene with the sperm nuclei at room temperature for about 20 minutes such that the DNA transgene is taken up by the sperm nuclei, providing mature zebrafish eggs, injecting the incubated sperm nuclei into the mature eggs using an injection needle having an outside diameter of about 10 to about 15 μm, and discarding injected eggs which exhibit no cleavage or abnormal cleavage. In yet at further embodiment, the invention provides transgenic teleosts and zebrafish produced according to the methods described above.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows demembranated zebrafj sh sperm nuclei in brightfield image showing sperm heads (Figure 1A) and in fluorescent image showing Hoechst L (# 33258) fluorescent dye labeling of nuclei (Figure IB) . Figure 2 is a schematic diagram of the apparatus used for injection of teleost eggs.

Figure 3 shows a teleost egg with an injection needle near the icropyle (arrow) .

Figure 4 shows three different embryos produced by intracytoplasmic sperm injection at 3.5 hours (Figure 4A) , 6 hours (Figure 4B) , and 26 hours (Figure 4C) after fertilization by sperm injection.

Figure 5 demonstrates green fluorescent protein expression in embryos obtained by intracytoplasmic sperm injection 24 hours (Figure 5A) and 7 days (Figure 5B) after fertilization with sperm preinσubated with pESG. Both images are projections of multiple focal planes.

Figure 6 is a schematic illustration of plas id pXeX, a vector developed for expression in Xenopus embryos. Figure 7 shows the sequence of the EFlα promoter of

Xenopus laevis (SEQ ID NO:l).

Figure 8 provides a map of pCSGFP3.

BRIEF DESCRIPTION OF THE PREFERRED EMBODIMENTS The methods of this invention allow transgenic fish with non-mosaic expression σf the transgene to be produced reliably by intracytoplasmic sperm injection. The method can be used in species which have small eggs because a small bore needle (less than about 15 μm in outside diameter) is used to inject the sperm into the eggs. The small bore needle can be used in this method because it uses sperm which is non-decondensed in which Lhe transgene is incorporated during a preincubation.

The sperm-mediated transgenesis of this invention allows misexpression experiments to be carried out more efficiently, without the need for keeping a line of transgenic animals. The non-mosaic expression provided by these methods is especially important for experiments involving genes that must be expressed in all cells of a particular tissue or body region, for example in studies of patterning of the embryonic axis or brain in early development- In addition, sperm-mediated transgenesis provides a means of generating large numbers of transgenic fish with different recombination events, an invaluable tool in the generation of fish with targeted recombination.

If non-decondensed sperm is incubated with a lineari∑ed DNA containing a transgene for about 20 minutes prior to injection, transgene expression can be obtained in all cells of the embryo. Nuclei do not have to be decondensed for expression, unlike methods used in the prior art. Short incubation periods, however, are insufficient to result in non- mosaic expression of the transgene (incorporation of the transgene into the germ line) . Non-mosaic expression of a transgene according to the methods of this invention requires prolonged incubation of the exogenous DNA with the sperm nuclei prior to injection. This suggests that some form of association must occur between the DNA and sperm nucleus prior to fertilization, so that the transgene is retained in all nuclei. In general, incubation periods of about 15 minutes or longer at room temperature are required for non-mosaic expression of foreign DNA in the inventive method. Preferably, incubations of about 15 to about 50 are used, while 20 minute incubations are most preferred.

The use of condensed sperm nuclei allows injections to be made with a small bore pipette injection needle, which is important for successful injection of relatively small eggs without cytoplasm leakage or other damage. Non-decondensed sperm nuclei generally have a diameter of approximately 5 μm. This technique is more efficient for generating non-mosaic transgenic fish, such as zebrafish, compared to injection of the foreign DNA into fertilized eggs, and is applicable to other teleosts such as medaka or any oLher desired teleost. The method is particularly suited for species with smaller eggs, however the methods may be used with other species such as Xenopus laevis and X. tropicalis, where sperm-mediated transgenesis currently is carried out with decondensed sperm nuclei and restriction enzyme mediated integration. Any desired transgene may be incorporated into the germ line of the transgenic fish. Examples of transgenes which may be useful include, for example, growth factor receptors. Reporter genes such as green fluorescent protein also may be used. Persons of skill in the art are capable of creating suitable constructs for insertion of any desired DNA into transgenic fish using the methods of this invention.

Demembranation may be achieved by any suitable method known in the art. For example, freeze-thaw methods, or the lysolecithin exposure methods described by Kroll and Amaya (4), which is hereby incorporated by reference. To check the sperm for demembranation, damage to the plasma membrane may be inferred from two observations. First, the tails of demembranated sperm can not be seen under a compound microscope. Untreated sperm have clearly visible tails. Second, only a minority of sperm nuclei are labeled by 1 μg/ l Hoechst dibenzimide B2883 (Sigma) prior to demembranation whereas after demembranation, all nuclei are fluorescent. See Figure 1.

To establish the optimal conditions for injecting sperm, the nuclei may be diluted about 1:20 in an appropriate buffer, such as sperm dilution buffer (see Example 1 for contents), labeled with Syto 11 (Molecular Probes) and loaded into capillaries. A series of injections may be performed onto a glass slide, and the injection volume adjusted such that each injection provides an average of one sperm nucleus. The injections were performed using glass injection needles pulled from thin-walled capillaries. Any capillary which can produce a needle of suitable diameter may be used, however, capil] aries having an outside diameter of about 1 mm and an inside diameter of about 0.8 mm are preferred. The capillaries a r<= pulled to produce a needle having an outside diameter of about 10 μm to about 15 μm, or preferably about 10 μm to about 12 μm. Most preferably, the outside diameter of the injection needle is about 10 μm. Inside diameters must be large enough to accommodate the size of the sperm nuclei being injected. For zebrafish, an inside diameter greater than about 5 μm is necessary. Preferably, the inside diameter is about 6 μm to about 8 μm.

Using these glass injection needles and traditional aqueous buffers, nuclei, settled and attached to the surface of the glass after a period of time. As a result, sperm injections could no longer be performed approximately 10 minutes after filling the injection needle. Coating the capillaries with Sigmacote^rrf or gelatin did not entirely solve the problem and sperm nucleus flow eventually stopped. To reduce this problem, the nuclei were injected as a suspension in a viscous buffer containing 6% polyvinylpyrolidone. The increased viscosity of this medium prevented nuclei from settling, and a reliable rate of nuclear inject..on could be obtained for over an hour. The use of a viscous injection buffer also protects the nuclei from shearing during infection through the smaller needle and allowed small and consistent injection volumes to be obtained using standard gas-pressure injectors and capillaries with 10 μm openings. Therefore, a more viscous medium is preferred. Any suitable and compatible medium of sufficient viscosity may be used with this method. However, if only a short time s needed for injection, ordinary aqueous medium may be used.

Injections preferably are made into the animal pole region of eggs. Removal of the choπoπ is unnecessary, and was m fact found to lead to premature egg activation. Eggs may be easily orientated and penetrated using needles with a tip of 10 to 15 μm (outer diameter), when held in agarosc wells or troughs and immersed in saline. See Figure 2- A relatively high salt buffer, such as Hanks saline, is preferably used, however any buffer compatible with the eggs is suitable. Any convenient method of injection may be used, however small needle diameter (about 15 μm) is important to the success of the method since larger bore needles may damage smaller teleost eggs. The inside diameter of the needle must be of sufficient size to allow the passage of the sperm nuclei.

After injection, eggs may be transferred to dishes containing E3 (5 M NaCl, 0.17 mM KCl, 0.33 M CaCl,, 0-33 mM Mg≤O , or any suitable medium (with minimal transfer of the high salt" (Hanks) buffer used in the injection apparatus. This method results in activation with minimal leakage of cytoplasm from or damage to the eggs. Eggs that cleave normally can develop into swimming larvae and give rise to fertile adults expressing the transgene. See Figure 5. All cells of the embryos pictured appear to express the marker gene, green fluorescent protein (GFP) . Clusters of brighter cells are visible (arrows); presumably these contain extra copies of the transgene. Seven days after fertilization the cells of the embryos were still expressing the marker gene. See Figure 5B.

EXAMPLE

Example 1. Transgenesis by Fertilization of Zebrafish Eggs.

Testes were dissected from 4 adult zebrafish males which had been killed by immersion in iced water. Testes (located on either side of the swim bladder) were removed with fine forceps. Demembranated sperm nuclei were prepared essentially as described by Kroll and A aya (4) - The demembranated sperm nuclei were quick frozen in liquid nitrogen in aliquots of 10 μl, at a concentration of approximately 100 nuclei/nl and stored at -80°C. Alternatively, nuclei were demembranated by freeze- thawing, were washed twice in 9 ml nuclear isolation medium (NIM; 123 M KCl, 2.6 mM NaCl, .8 mM NaH₂PO„, 1.4 mM KH,P0„, 3 mM EDTA, 0.5 mM PMgF, pH 7.2) (7) with 5% BSA, washed once in 1 ml NIM, and finally resuspended in 250 μl NIM. The concentration of sperm nuclei was checked by counting nuclei labeled with Hoechst #33258 or Sytoll (Molecular Probes) dyes in a hematocytometer. See Figure 1 (Hoechst #33258) .

Sperm aliquo s were thawed on ice, and then mixed by pipetting up and down through a small pipette tip. An aliquot of 2.5 μl was transferred to an Eppendorf™ tube, and 0.5 μl linearized DNA (35 ng either pESG or pB0S-H2GFP) was added (concentration during incubation was 0.3 ng/ml) . pESG contains green fluorescent protein (GFP) driven by the Xenopus EFlα promoter, cloned into pCSGFP3 (see Figures 6-8) . pB0S-H2GFP (Phar ingen, cat. no. 40011P) contains eGFP fused to histone, also regulated by the Xenopus EFlα promoter. The mixture of sperm nuclei (100 nuclei/nl) and DNA was incubated at room temperature (23°C - 25'C) for 20 minutes, then diluted with sperm dilution buffer (SDB; 250 mM sucrose, 75 mM KCl, 0.5 mM spermidine trihydrochloride, 0.2 mM spermine tetrahydrochloride, pH 7.3) (4) or MOH buffer (10 mM κpo_<, pH 7.2, 125 mM potassium gluconate, 5, mM NaCl, 0.5 mM MgCl,, 250 mM sucrose, 0.25 mM spermidine, 0.125 mM spermine) (5) containing polyvinylpyrolidone (6% final concentration; Sigma 5288) . The final volume of diluted sperm solution was 50 μl,

Female zebrafish were anesthetized with tricaine (Sigma A5040), placed on a clean piece of parafilm in a petri dish, and gently squeezed to expel mature eggs. Eggs were kept in a mound, and the dish immediately covered to prevent dehydration. A Pasteur pipette was used to transfer eggs (approximately 40 at a time) to the injection apparatus- This consists of v-shaped troughs cut into 1.2% agarose in Hanks saline. See Figure 2. The troughs were filled with Hanks saline to a level so that the eggs were just immersed, with less than 1 mm distance between the top of the eggs and the bottom of the meniscus, to ensure efficient withdrawal of the injection needle from eggs.

Injection needles were made by pulling thin-walled capillaries (Clarks GC100T; 1.00 mm OD and 0.78 mm ID prior to pulling) on a Flaming-Brown puller (Sutter; heat = ramp value -5, pull = 75, velocity «= 25, time = 50), and breaking the tips with forceps so that the outer diameter of each needle was 10 to 15 μm. Sperm nuclei were backfilled with 3 μl of the sperm nuclei suspension into a two centimeter length of Tygon™ tubing (AAC00001) using a 20 μl pipetter with a small pipette tip attached. The needle was then inserted into the tip of the tube. The pipette tip was removed from the pipetter and attached to a 200 μl pipetter. This pipetter was used to force the sperm suspension into the tip of the glass injection needle. Injections were carried out with a gas pressure injector (Narishige IM 3000 or Medical systems Corp. PLllOO; 3.0 psi; 100 msec) . Needles were mounted on a holder on a Leica mechanical manipulator, and injections were done under a dissecting microscope (Leica MZ12), with a transmitted-light base. Sperm nuclei were visible under the dissecting microscope using darkfield illumination. Sperm nuclei were injected into the animal pole region of the egg- Eggs were penetrated about 50 to 100 μm from the micropyle, which is visible under brightfield illumination, then rotated so that the tip was near the micropyle prior to injection. After a batch of eggs were injected, they were transferred with a Pasteur pipette to a large petri dish containing E3 and then placed in a 28.5°C incubator. One hour after activation, the eggs were checked and normally cleaving embryos were selected and grown at 28.5°C. Non-cleaving eggs and embryos with abnormal cleavage were removed. Approximately 150 to 200 eggs were injected in an hour with this technique. Out of 20 eggs that cleaved normally, 12 were morphologically normal after 24 hours. See Table ϊ and Figure 4. Embryos that had more than two cells after first cleavage, indicating polysper y, were retarded in epibσly and failed to develop normally. Embryos that appeared normal at 24 hours were maintained until adulthood. Eight out of 12 survived, and when crossed to one another, the survivors gave rise to normal embryos. Table I: Injection of Sperm Nuclei into Zebrafish Eggs

Results of 4 different injection sessions using nuclei that were demembranated by lysolecithin treatment (a) or freeze-thawing (b).

When DNA was incubated with sperm nuclei for 20 minutes at room temperature prior to dilution in SDB, almost all (54 out of 56) early embryos were GFP-positive. Fifteen out of 56 embryos were completely green at 24 hours post fertilization. Confocal microscopy indicated that all cells expressed GFP. Some cells, usually in clusters, were brighter than others. See Figure 5A. Fluorescence also was detected in embryos at 7 days post fertilization, apparently in all cells, with some cells still appearing brighter than others. See Figure 5B. When DNA was incubated with sperm nuclei for 5 minutes prior to dilution in SDB, no GFP-positive embryos were obtained (data not shown) . Also, when embryos obtained by normal fertilization were injected with the same DNA at a concentration of 0.7 ng/μl, which is the effective concentration in the injection buffer, no GFP-positive cells were seen (data not shown) .

REFERENCES 1. Stuart, G. ., McMurray, J. V. & Westerfield, M, Replication, integration and stable germ-line transmission of foreign sequences injected into early zebrafish embryos. Development 103: 403-412 (1988).

2. Raz, E., van Luenen, H. G., Schaerringer, B,, Plasterk, R. H. A. & Driever, . Transposition of the nematode Caenorhabditis 3. Linney, E., Hardison, N- L., Lonze, B. E., Lyons, S. & DiNapoli, L. Transgene expression in zebrafish: A comparison of retroviral-vector and DNA-injection approaches. Development Biology 213: 207-216 (1999),

4. Kroll, K. L. & Amaya, E. Transgenic Xenopus embryos from sperm nuclear transplantations reveal FGF signaling requirements during gastrulating. Development 12:, 3173-3183 (1996).

5. Offield, M. F., Hirsch, N. & Grainger, R. M. The development of Xenopus tropicalis transgenic lines and their use in studying lens developmental timing in living embryos-. Development 127: 1789-1797 (2000).

6. Perry, A, C. et al. Mammalian transgenesis by intracytoplasmic sperm injection. Science 284: 1180-1183 (1999) .

7. Kuretake, S., Klmura, Y., Hoshi, K. & Yanagimachi, R. Fertilization and development of mouse oocytes injected with isolated sperm heads. Biol . Reprod. 55: 789-795 (1996).

8. Chan, A. W. et al. Foreign DNA transmission by ICSI: injection of spermatozoa bound with exogenous DNA results in embryonic GFP expression and live rhesus monkey births. Molecular Human Reproduction 6: 26-33 (2000) .

Claims

1. A method of producing a fertile transgenic teleost which comprises :

(a) providing a suspension of demembranated sperm nuclei wherein said sperm nuclei are not decondensed; (b) providing a DNA transgene;

(c) incubating said DNA transgene with said sperm nuclei under conditions such that said DNA transgene is taken up by said sperm nuclei ,-

(d) providing mature eggs of the same species as said sperm nuclei;

(e) injecting said incubated sperm nuclei into said mature eggs, wherein said injection is performed using an injection needle having an outside diameter of about 10 to about 15 μm; and

(f) discarding injected eggs which exhibit no cleavage or abnormal cleavage.

2. A method of claim 1 wherein the conditions of incubation include room temperature incubation for about 15 to about 50 minutes .

3. A method of claim 1 wherein conditions of incubation include room temperature incubation for about 20 minutes.

4. A method of claim 1, 2 or 3 wherein said injection needle has an outside diameter of about 10 μm to about 12 μm.

5. A method of claim 4 wherein said injection needle has an outside diameter of about 10 μm.

6. A method as claimed in any one of the preceding claims wherein said injection needle has an inside diameter of about 6 μm to about 8μm.

7. A method as claimed in any one of the preceding claims wherein said teleost is a zebrafish.

8. A method of producing a fertile transgenic zebra ish which comprises : (a) providing a suspension of demembranated zebrafish sperm nuclei wherein said sperm nuclei are not decondensed;

(b) providing a DNA transgene;

(c) incubating said DNA transgene with said sperm nuclei under conditions such that said DNA transgene is taken up by said sperm nuclei, wherein said conditions include room temperature incubation for about 20 minutes;

(d) providing mature zebrafish eggs;

(e) injecting said incubated sperm nuclei into said mature eggs, wherein said injection is performed using an injection needle having an outside diameter of about 10 to about 15 μm; and

(f) discarding injected eggs which exhibit no cleavage or abnormal cleavage.

9. A transgenic teleost produced according to any one of claims 1 to 7.

10. A transgenic zebrafish produced according to claim 8.