WO2002088368A1

WO2002088368A1 - Spatial and temporal control of gene expression in the zebrafish using baculovirus

Info

Publication number: WO2002088368A1
Application number: PCT/SG2001/000079
Authority: WO
Inventors: Mahendra Dinkar Wagle; Suresh Jesuthasan
Original assignee: Institute Of Molecular Agrobiology
Priority date: 2001-05-02
Filing date: 2001-05-02
Publication date: 2002-11-07
Also published as: TW200407432A

Abstract

This invention provides a method for expressing genes in zebrafish in a spatially and temporally controlled manner using baculovirus. The method allows study of genes in particular preselected locations or cells during early or late stages of development without the need for producing transgenic animals.

Description

SPATIAL AND TEMPORAL CONTROL OF GENE EXPRESSION IN Tiffi ZEBRAFISH USING BACULOVIRUS

BACKGROUND OF THE INVENTION

1. Technical Field

This invention relates to the field of molecular biology, and to methods for controlling gene expression spatially and temporally. In particular, the invention relates to such gene 5 expression control in zebrafis using baculovirus.

2. Description of the Background Art

The zebrafish has become an established model organism in developmental biology primarily because of its use as a system

L0 for the study of expression and functions of genes. It is particularly useful because it is a simple biological system which has a high degree of genetic similarity to the human genome .

To complement loss-of-function data provided by zebrafish

L5 mutants, gene misexpression experiments often are carried out in addition. This is usually accomplished by injection of mRNA of the gene of interest at the single cell stage. This approach generally is not useful to study events in gene expression which occur later in development, for example topographic map

_0 formation in the visual system or axon guidance in the olfactory system, since when the mRNA is injected at an early stage of development there is a chance that the mRNA will interfere with earlier functions, masking the effects it is desired to study. Additionally, there is a greater chance that the mRNA will be

25 degraded by the time it is reguired. Therefore, a system that allows expression of a gene at specific later time points in development, and particularly in a small subset of cells which are desired to be studied, would be highly useful for investigating late processes such as those mentioned above.

Viruses have been used as a misexpression tool in other model organisms such as the chick. Replication-incompetent 5 viruses, for example RCAS, have been used to study brain patterning, axon guidance and vasculogenesis (1-31) . Sheer and Campos-Ortega have demonstrated that the Gal-4/UAS system can be used to drive gene expression in zebrafish (19) . The Gal4 system has the advantage that expression always occurs in a

L0 specific set of cells, dependent on the expression pattern of

Gal4; multiple embryos with identical patterns of overexpression can be analyzed. A drawback of this system, however, is that stable transgenic lines must be established in order to use it. Another recently proposed system for ectopic gene expression,

L5 the HSP70 promoter combined with laser heating to induce expression in cells of interest (9) , also requires the production of stable transgenic lines, greatly increasing the cost and time involved.

An alternative method for localized misexpression late in

-0 development is mRNA injection by iontophoresis into the cells of interest (20) . This method of injection is relatively difficult, however. Viral injection methods are performed by injecting a viral vector into the extracellular spaces of the desired tissue using standard gas-pressure injectors or any

.5 other suitable injectors. Viral systems enable ectopic expression in the injected animal, and thus provide a faster test of gene misexpression. A retrovirus which has been used for this method in zebrafish is the pseudotyped Moloney urine leukemia virus (21, 22) . This retrovirus acts as a mutagen, as

30 it can integrate into the genome, and provides fairly long-term expression of an exogenous gene. Production of the pseudotyped retrovirus is cumbersome compared to other viruses, however, and the maximum insert size is fairly small. More importantly, retrovirus can infect only dividing cells. Methods dependent on

35 retroviral vectors therefore are not as useful for studies in cells at later stages of development or for studies of all cell types .

Baculoviruses are double stranded DNA viruses, and are thought to be taken up by endocytosis (17) . Once inside a cell, 5 baculovirus DNA usually remains episomal (18) . They now are known to infect human, murine, Xenopus, and Drosophila cells, including both dividing and non-dividing cells. Baculoviruses are able to replicate only in insect cells, however (4-8). In general, baculovirus can accommodate inserts of exogenous DNA up

L0 to 15 kb in length.

A simple and effective method which enables the performance of misexpression assays in a preselected location and at a desired time, accommodates large gene inserts and is useful at all stages of development is needed in the art.

L5

SUMMARY OF THE INVENTION Accordingly, this invention provides a method of expressing at least one gene in specific preselected cells of a zebrafish

20 which comprises injecting into the extracellular space around said preselected cells or their developmental precursors in a zebrafish embryo a baculovirus carrying said gene. The method is useful for cells which are terminally differentiated, and can be used for the expression of one or more genes. Preferred

25 methods involve those in which a first gene and a second gene are expressed, wherein the first gene is a reporter gene such as green fluorescent protein and the second gene is a gene the expression of which is to be studied in said preselected cell type. The second gene may be put under the control of an

30 inducible promoter such as a heat shock protein promoter. Preferred methods involve the injection of high titer baculovirus at a concentration of at least 10¹⁰ particles/ml, preferably of about 10¹⁰ to about 10¹¹ particles/ml, and most preferably of about 10¹¹ particles/ml.

35 In further embodiments, the invention provides a misexpression assay which comprises systematically expressing a library of genes in specific preselected cells of a zebrafish according to the methods describe above.

In yet further embodiments, the invention provides zebrafish expressing at least one gene in specific preselected 5 cells according to a methods described above.

In yet further embodiments, the invention provides a method of reducing or eliminating expression of a gene in specific preselected cells of a zebrafish which comprises injecting into the extracellular space around said preselected cells or their

L0 developmental precursors in a zebrafish embryo a baculovirus carrying an antisense oligonucleotide which binds to said gene. In yet further embodiments, the invention also relates to the use of a baculovirus vector for expression of a gene in zebrafish.

L5 The invention further provides a baculovirus vector which comprises a reporter gene driven by a ubiquitous promoter and a second expression cassette containing a ubiquitous or inducible promoter, a multiple cloning site and a polyadenylation sequence. The invention also provides a. kit for expression of a

10 gene in specific preselected cells of a zebrafish which comprises a baculovirus vector as described above further containing a second promoter which is a tissue-specific or cell type-specific promoter, such as HuC, or an inducible promoter such as the Hsp70 promoter. The invention also provides a kit

25 for expression of a gene in specific preselected cells of a zebrafish which comprises a baculovirus vector and at least one construct containing a cell type- or tissue-specific promoter, a multiple cloning site and a polyadenylation sequence.

30

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a series of photographs showing widespread or localized gene expression in zebrafish embryos. Figure 1A shows a two-day embryo (lateral view) with widespread expression of 35 the marker gene nβGal. Figure IB shows localized expression of the marker gene green fluorescent protein (GFP) in the eye (lateral view) . Figure 1C shows neuronal tracing in the visual system with unc76-GFP (dorsal view) . All embryos are oriented with anterior to the left.

Figure 2 is a series of photographs showing simultaneous expression of two genes by one virus in zebrafish embryos injected with baculovirus vector into the posterior eye primodium of 18-20 somite stage embryos. Figures 2A and 2C show embryos imaged by confocal microscopy for GFP expression prior to X-Gal staining. Figure 2B shows expression of β-Gal, induced 24 hours after virus injection. Figure 2D shows embryos incubated at 28°C for 30 hours after virus injection.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS This invention pertains to a method of expressing a gene or genes in a preselected location or in preselected cells of a zebrafish by injecting a baculovirus vector harboring the gene(s) into the extracellular space surrounding the preselected cells or their developmental precursors. This method permits the study of expression or misexpression of a particular gene or genes in a particular location, tissue or cell type in the zebrafish at any desired stage of development. Any gene or genes may be expressed, including antisense oligonucleotides which can bind to a specific gene to inhibit expression. Thus, the method can be used to study both the effect of a misexpressed gene and to create "knockout" animals which do not express a particular gene in a specific tissue or location.

The methods described and exemplified here rely on the unexpected ability of baculovirus, specifically Autographa californica multiple nuclear polyhidrosis virus (AcMNPV) , to infect zebrafish embryos and reliably drive expression of any desired gene in vivo. Reporter genes such as GFP and LacZ, and any exogenous gene may be studied by misexpression or knocked out in one or a variety of preselected cell types. Previously, spatially and temporally controlled gene expression in zebrafish has been difficult to obtain without having to produce transgenic animals.

Baculovirus was thought to infect only insect cells. It has been discovered recently that it may also infect some other 5 species as well, but baculovirus has not been used in teleosts. Surprisingly, the studies reported here reveal that baculovirus carrying an exogenous gene can infect, upon injection into a specific site in zebrafish embryos, a variety of zebrafish cells and drive gene expression in vivo. This technique can be used to

L0 transiently misexpress one or more exogenous genes in desired cells for assay of the gene products' effects on those cells. By misexpression it is meant that a gene is expressed in a location where or at a time when it is not normally expressed. This technique therefore allows the function of genes to be tested in

L5 specific tissues, even late in development. Assay of libraries of genes in a single preselected tissue or area may be performed using this method, or a single gene may be studied in many different locations. One or many genes may be studied at different times in development in the same way. Baculovirus also

20 may be used in these methods to express an antisense which inhibits or eliminates the expression of an endogenous gene in any desired location or stage in development.

The disclosures of this specification are intended to refer to all of these methods. For convenience, the term "gene" or

25 "exogenous gene" is intended to signify any genetic material, including genes or portions of genes, nucleotides or oligonucleotides including antisense or ribozyme sequences or any DNA not normally present in the baculovirus vector. The term is intended to include genetic material from any source, including

30 zebrafish or any other species. Large constructs up to about 15 kb or larger, including double expression cassettes, can be made for expression using the methods of this invention.

To extend the usefulness of zebrafish as a model system for developmental biology, especially for the study of events late in

35 development, this method allows gene misexpression in a spatially and temporally-controlled manner which is efficient enough to make large scale screening of genes in specific cell types or stages of development practical. For example, it is possible to search for genes involved in topographic map formation by injecting pools of virus containing genes from a retinal ganglion 5 cell library and a neuronal tracer into specific regions of the eye. This approach can potentially uncover genes that would be overlooked in typical loss-of-function screens.

By microinjecting high-titer baculovirus into specific locations of zebrafish embryos, such as, for example, different

L0 regions of the eye, the position of expression can be controlled. Using a heat-shock promoter or any other convenient inducible promoter, the time of expression can also be controlled. Using a high titer of virus is key to the success of this method. Concentrations of at least 10¹⁰ particles /ml should be used,

L5 however it is preferred to use about 10¹⁰ to about 10¹¹ particles/ml and most preferably about 10¹¹ particles/ml.

Baculoviruses can be engineered to express two or even more genes under independent promoters, allowing cells to be labeled by a reporter gene such as GFP while the gene of interest being

20 tested is simultaneously expressed within the embryo.

Baculovirus carrying a marker gene such as GFP, driven by Xenopus EFI promoter or the endogenous polyhedrin promoter, at a titer of about 1 x 10¹¹ particles/ml is preferred for use in these methods. The EFIα promoter is known to drive expression in all

25 cells when DNA is injected in plasmid form at the one cell stage, however any convenient promoter may be used depending on whether constitutive, inducible or other expression of the gene is desired. Suitable promoters include, for example the zebrafish HuC promoter (see Park et al., Dev. Biol. 227 (2) : 279-293, 2000)

30 and the human cytomegalovirus immediate early promoter. Any marker gene which is convenient for the assays being used is compatible with the invention. For example, the marker genes encoding green fluorescent protein, LacZ or any marker gene may be used. Viruses should be concentrated to a high titer for

35 injection into zebrafish embryos. To simultaneously label and manipulate a cell, a marker gene such as GFP should be co-expressed with the gene being tested. This allows infected cells to be identified. GFP also allows the infected cells to be imaged, if desired. Using a baculovirus 5 vector, multiple genes can be expressed from one virus. Methods using a separate promoter for each gene are preferred, however any suitable method for coexpression of two or more genes may be used. For example, a double expression cassette or an internal ribosomal entry site (IRES) may be used. A dicistronic

L0 expression cassette, in which, for example, tauLacZ or tauGFP is coexpressed with the gene of interest under the EFIα promoter and an EMCV IRES is suitable. The gene of interest is advantageously expressed under the control of an inducible promoter such as, for example the zebrafish Hsp70 promoter. The

L5 heat shock protein promoter is most preferred due to the ease of induction in vivo in the zebrafish.

Insertion of genes into baculovirus may be done by any convenient prior art method. Generally, a transfer plasmid is constructed by suitable methods known in the art for transfer of

20 the desired construct into the baculovirus, however any methods can be used. Those possessing ordinary skill in the art of genetic manipulation are well aware of many methods to achieve the desired transfer of any genetic material to baculovirus. The examples below provide a suitable method for construction of

25 transfer vectors and expression cassettes which are useful, however skilled molecular biologists are able to modify these methods to achieve any desired result. Production of suitable virus stocks is likewise known in the art, and any of the known methods are contemplated for use in these methods. Insertion of

30 the genetic material may be performed advantageously as described in Example 1, by cotransfection of transfer plasmid and linearized baculovirus genome into SF9 (insect) cells, although any convenient method may be used.

An alternative method (the bac-to-bac method) allows

35 production of virus in a shorter time. This method involves transformation of E. coli strain ^"DHlObac, which carries the baculovirus genome, with transfer plasmid. Genomic DNA from infected colonies may be prepared according to known methods and used for transfecting SF9 cells. Virus may be purified and amplified by any suitable method and concentrated. Viruses are 5 preferably further purified before injection.

Most baculovirus systems require picking a single colony, either by plaque purification or end-dilution, and subsequent amplification, which can take up to a month. The bac-to-bac system, however, allows production of virus within 2 weeks.

.0 Also, with this system, large number of different virus constructs can be made, for example from a normalized library, in a simpler manner than with conventional techniques. This allows misexpression screens to be carried out for a large number of genes in an efficient manner.

.5 Injection of the baculovirus into the zebrafish embryo preferably is performed using a gas pressure icroinjector, such as those obtainable from Medical Systems Corporation. It is convenient to aspirate concentrated virus particles into the tip of injection needles. Appropriate needles may be pulled from

!0 thin wall borosilicate capillaries according to known methods to achieve a needle of the desired diameter.

Embryos may be injected at the sphere stage or in later stages, such as, for example, the 8 somite, 12 somite or 20 somite stages or later, as desired. The cells exposed to virus

25 in the extracellular space take up the virus and become infected. Therefore the site and volume of the injection can be used to control the cells which are exposed. For example, infectious particles may be injected into the eye of a zebrafish embryo after the bud has formed. Infected cells are scattered only in

30 the eye after this type of injection, so that the role of the gene of interest may be studied in that tissue. The methods can be used to study the consequences of expression of any gene in any tissue of the zebrafish at early or late stages of development. Injections may be made into the area around 5 developmental precursors of the cells desired to be studied as well^". For convenience, the invention provides kits for the expression of genes in zebrafish. These kits contain a baculovirus vector which comprises a reporter gene such as GFP driven by a ubiquitous promoter and a second expression cassette 5 with a ubiquitous or inducible promoter followed by a multiple cloning site and a polyadenylation signal sequence. The construct may contain, for example, a cell type- or tissue- specific promoter such as HuC, a multiple cloning site and a polyA signal. This type of construct may be used to misexpress .0 genes in neurons. Kits according to the invention also may further comprise a second construct containing a promoter active in zebrafish, a multiple cloning site, a yc-tag and a polyadenylation sequence.

The following examples are included to illustrate certain L5 embodiments of the invention and are not intended to be limiting in any way.

EXAMPLES

Example 1. Constructs. 20

A transfer plasmid (pGTV) containing green fluorescent protein (GFP) , under the control of the Xenopus EFIα promoter, was constructed by cloning a blunted DralllNotl fragment of pESG into pAcSG2 (Pharmingen) . To create a second expression

25 cassette, the hsp70 promoter, as described in reference 9, was cloned into the Notl/Kpn site of pGTV. The HSV-TK poly' A signal was amplified from pCEP4 (Invitrogen) by PCR and cloned into the Apal/Kpnl sites to generate the pGHT transfer plasmid. A blunted Hindlll/Stul fragment of pCS2+nβgal, as described in reference

30 10, was cloned into the blunted Apal site of pGHT . The transfer plasmid pGHT-Pp-nβGal was generated from pGHT by removing the polyhedrin promoter.

A transfer plasmid containing GFP under the endogenous polyhedrin promoter (pAC-GFP) was constructed in two steps. 5 First, the Ncol/Stul fragment of pEGFP (Clontech) was cloned into the Ncol/S al site of pAcSG2. The Xbal/Bglll SV40-poly A fragment of pXeX, as described in reference 11, was cloned into the Xbal/Bglll site of pAcSG2+EGFP to generate pAC-GFP.

A blunted EcoRI/Xbal fragment of IRES-tau-GFP-LNL, as described in reference 12, was cloned into the blunted Clal site of modified pXeX. A Xhol/Bglll fragment of this was cloned into the pAcSG2 Xhol/Bglll sites to construct pACSG2-EITG. A blunted Hindii/SnaBI fragment of pCS2~NLSβGal was cloned into the Pmel site of pACSG2-EITG to generate pACSG2-EITG-nβGal .

A transfer plasmid pFB-Unc76-GFP was constructed by cloning the Xhol/Notl fragment of XEX-76/eGFP, as described in reference 13, into the Sall/Notl site of pFastBac Dual (Gibco-BRL) . See Table I, below.

One microgram transfer plasmid was cotransfected with 100 ng linearized baculovirus genome (BacPAKβ; Clontech) into SF9 cells using Transfecting (Clontech) . See Table II, below.

Table I. Constructs.

Table II Viruses ,

Example 2. Virus Production and Microinjection of Zebrafish Embryos .

Viral plaques were purified and amplified as described by King and Possee (14) . SF9 cells infected by recombinant virus expressed GFP under control of the EFIα promoter, allowing identification of positive clones. Viruses were amplified in several 175 cm³ tissue culture flasks to obtain at least 100 ml virus suspension in culture medium. After amplification, virus particles were concentrated by sedimentation at 22,000 rpm at 4°C for one hour in a Type-50.2 rotor (Beckman) . Viruses were further purified by banding on a 10-50% one step sucrose gradient in a SW28 rotor (Beckman) at 24,000 rpm at 4°C for one hour. Sucrose was removed to obtain purified viruses by diluting with O.lx TE (10 mM Tris HC1, 1 mM EDTA, pH 8.0) and concentrated again by sedimentation at 22,000 rpm at 4°C for one hour in a Type 50.2 rotor (Beckman). The final virus pellet was resuspended in 10 μl Hanks saline (0.137 M NaCl, 5.4 mM KCl, 0.25 mM Na₂HP0₄, 0.44 mM KH₂P0₄, 1.3 mM CaCl₂, 1.0 mM MgS0₄ and 2 mM NaHC0₃) . Concentrated virus stocks were kept at 4°C and used for injections for at least three months with no discernable loss of infectivity.

Alternatively, virus was produced by the bac-to-bac method (15) , which allows production of virus in a shorter time. Recombinant viruses were generated according to the bac-to-bac -manual (Gibco-BRL) , which is hereby incorporated by reference. Briefly, transfer plasmid was transformed into E. coli strain DHlObac, which carries the baculovirus genome. Colonies carrying recombinant virus (white colonies on X-gal containing plates) were selected. Genomic DNA prepared from a selected colony was used for transfecting SF9 cells. After four days of incubation of the transfected cells at 28°C, supernatant containing virus was collected and used for further amplification. Viruses were concentrated and purified as described above. Concentrated virus particles (lxlO¹¹ particles/ml) were aspirated into the tip of injection needles pulled from thin wall borosilicate capillaries (Clarks GC100TF) and injected into the zebrafish embryos using a gas pressure microinjector (PLI-100; Medical Systems Corp.). For injection at the sphere stage, embryos with chorions were kept on a glass slide with minimum egg water (E3) in a cluster of 10-15 embryos. For injections at 12- somite and later stages, individual dechorionated embryos were embedded in 1.2% low melting temperature agarose (BioRad) for injection.

Example 3. Xgal Staining and Microscopy.

Embryos injected with viruses carrying the nβgal construct were stained as described by Muller et al . (16) . Embryos were washed in sodium phosphate buffer and fixed in fixing solution

(100 mM phosphate buffer, pH 8.0, containing 2 mM MgCl₂, 5 mM EGTA and 1% glutaraldehyde) for 2 hrs at 37°C. Embryos then were rinsed two times in washing solution (100 mM phosphate buffer, pH 8.0, containing 2 mM MgCl₂, 0.03% NP40 or IGEPAL CA-630, 0.01% Na-deoxycholate) . Fixed embryos were incubated in washing solution containing 1 πig/ml X-gal, 25 mM K₃Fe(CN)₆ and 25 mM K₄Fe(CN)₆ for 2 hrs at 37°C.

Embryos stained with X-gal were mounted in 70% glycerol in PBS and imaged with a compound microscope using a color CCD camera. For imaging live embryos, the embryos were embedded in 1.2% low melting temperature agarose. Embryos expressing GFP were observed and photographed using an Axioskop (Zeiss) with an MRC 1024 confocal microscope (Bio-Rad) .

When baculovirus vector bearing the GFP gene was injected into zebrafish embryos at the sphere stage, widespread expression of GFP was seen after several hours. Out of 36 embryos injected, 21 survived and 9 showed GFP expression. In these, GFP was still present 5 days after injection. Similar widespread expression was seen when virus containing nuclear-localized LacZ under control of the Hsp70 promoter was injected into sphere stage embryos. See Figure 1A, which shows a two-day embryo with widespread expression of nβGal. This embryo had been injected with virus carrying HSP70-nβGal at 5 hours post fertilization (hpf) , and heat shocked 6 hours later. Results for zebrafish embryos injected with construct pAc-GFP, which carries GFP under the control of the polyhedrin promoter indicated that the endogenous polyhedrin promoter is weakly active in zebrafish (data not shown) . 5 When virus was injected into embryos at later stages of development, expression of the reporter genes was more localized. Expression to a specific region of the eye could be obtained by injecting virus into the eye primordia at 18 hours post- fertilization (18-20 somite stage) . Viruses carrying EFlα-GFP

L0 were injected into the posterior eye at the 18 somite-stage, and the embryo was imaged 24 hours later. See Figure IB. Expression of GFP was seen in a few somites when virus was injected into pre-somite mesoderm at the 8-10 somite stage. Localized expression in other tissues, such as the olfactory pit and brain,

L5 could also be obtained by injection at 40 to 48 hpf (data not shown) .

To test the ability of viruses to label axons of neurons in the visual system, baculovirus carrying GFP was injected into the animal pole region of embryos at 5 hpf. After three days, cells

20 in the eyes and brain were fluorescent. Although the cell bodies were green, fluorescent retinal ganglion cell and tectal axons were not detected. When a fusion of unc76-GFP was transferred to the embryos by a baculovirus vector, however, axons in the eye and brain were clearly visible. See Figure 1C, which shows that

25 terminal arbors of retinal ganglion cells (arrowhead) as well as dendrites of tectal neurons (arrows) , are clearly visible in the optic tectum of this 3 day old fish.

Example 4. Coexpression of Tracer and Marker Genes.

30

An internal ribosomal entry site (IRES) and a double expression cassette were each used to coexpress genes by plasmid injection at the 1-cell stage. The plasmid pACSG2-EITG- nβGal (expressing nuclear-localized β-gal with EFIα and tau GFP with an 35 IRES) or a double expression cassette bearing both GFP and LacZ, each under the control of a promoter, were injected. Expression of the plasmid pACSG2-EITG- nβGal resulted in only weak GFP fluorescence, while LacZ staining was intense. A virus containing GFP and LacZ under separate promoters resulted in strong expression of both reporters. This baculovirus vector, with constitutive GFP expression (with Xenopus EFIα promoter) and inducible nuclear βGal expression (with zebrafish HSP-70 promoter) , was injected into the posterior eye primodium of 18-20 somite stage zebrafish embryos. See Figure 2. Expression of LacZ was under heat shock control, and could be induced independently of GFP. Figures 2A and 2C show GFP expression prior to X-gal staining. In Figure 2B, expression of nuclear βGal was induced 24 hours after injection by incubating embryos at 37 °C for one hour. Embryos were left at 28°C for another 6 hours and then stained with X-gal. In Figure 2D, embryos were incubated at 28°C after virus injection for 30 hours and then stained with X-gal.

References

1. Logan et al., Rostral optic tectum acquires caudal characteristics following ectopic engrailed expression. Curr. Biol . 6(8) .1006-1014, 1996.

2. Friedman and O'Leary, Retroviral misexpression of engrailed genes in the chick optic tectum perturbs the topographic targeting of retinal axons. J. Neurosci . 16 (17) : 5498-5509, 1996.

3. Flamme et al., Overexpression of vascular endothelial growth factor in the avian embryo induces hypervascularization and increased vascular ^:permeability without alterations of embryonic pattern formation. Dev. Biol . 171 (2) : 399-414, 1995.

4. Barsoum et al, Efficient transduction of mammalian cells by a recombinant baculovirus having the vesicular stomatitis virus G glycoprotein. Hum . Gene Ther. 8 (17 ): 2011-2018 , 1997.

5. Merrihew et al . , Chromosomal integration of transduced recombinant baculovirus DNA in mammalian cells. J. Virol . 75(2) :903-909, 2001. 6. Oppeneimer et al . , Functional conservation of the wingless- engrailed interaction as shown by a widely applicable baculovirus misexpression system. Curr. Biol . 9 (22) : 1288-1296, 1999.

7. Sarkis et al., Efficient transduction of neural cells in vitro and in vivo by a baculovirus-derived vector. Proc. Natl . Acad. Sci . USA 97 (26) : 14638-14643, 2000. 8. Sandig et al., Gene transfer into hepatocytes and human liver tissue by baculovirus vectors. Hum . Gene Ther. 7 (16) : 1937-1945, 1996.

5 9. Halloran et al., Laser-induced gene expression in specific cells of transgenic zebrafish. Development 127 (9) : 1953-1960, 2000.

10. Fire et al., A modular set of lacZ fusion vectors for L0 studying gene expression in Caenorhabditis elegans. Gene 93(2) .189-198, 1990.

11. Johnson and Krieg, pXeX, a vector for efficient expression of cloned sequences in Xenopus embryos. Gene 147 (2) :23-26, 1994.

L5

12. Rodriguez et al., Variable patterns of axonal projections of sensory neurons in the mouse vomeronasal system. Cell 97(2): 199- 208, 1999.

20 13. Dynes and Nagai, Pathfinding of olfactory neuron axons to stereotyped glomerular targets revealed by dynamic imaging in living zebrafish embryos. Neuron 20 (6) : 1081-1091, 1998.

14. King and Possee, The Baculovirus Expression System A

25 laboratory Guide. (1992) Chapman and Hall. London, pp 107-126.

15. Luckow et al., Efficient generation of infectious recombinant baculoviruses by site-specific transposon-mediated insertion of foreign genes into a baculovirus genome propagated in Escherichia

30 coli. J. Virol . 67 (8 ): 4566-79, 1993.

16. Muller et al., Efficient transient expression system based on square pulse electroporation and in vivo luciferase assay of fertilized fish eggs. FEBS Letts . 324 (1) : 27-32, 1993.

35

17. Hofmann et al., Efficient gene transfer into human hepatocytes by baculovirus vectors. Proc. Na tl . Acad. Sci . USA 92(22) :10099-10103, ,1995. 0 18. Hartig et al., Insect virus: assays for viral replication and persistence in mammalian cells. J. Virol . Meth . 31(2- 3) -.335- 344, 1991.

19. Scheer and Camnos-Ortega, Use of the GA14-USA techniques for 45 targeted gene expression in the zebrafish. Mech . Dev. 80(2) :153-

158, 1999.

20. Dorsky et al., Control of neural crest cell fate by the Wnt signaling pathway. Nature 396 (6709) : 370-373, 1998.

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21. Linney et al., Transgenic expression in zebrafish: a comparison of retroviral-vector and DNA-injection approaches. Dev. Biol . 213 (1) : 207-216, 1999. 22. Lin et al., Integration and germ-line transmission of a pseudotyped retroviral vector in zebrafish. Science 265(5172) :666-669, 1994.

23. Park et al . , Analysis of upstream elements in the HuC promoter leads to the establishment of transgenic fish with fluorescent neurons. Dev. Biol. 227 (2 ): 279-293, 2000.

Claims

CLAIMS :

1. A method of expressing at least one gene in specific preselected cells of a zebrafish which comprises injecting into the extracellular space around said preselected cells or their developmental precursors in a zebrafish embryo a baculovirus carrying said gene.

2. A method of claim 1 wherein said specific preselected cells are terminally differentiated.

3. A method of claim 1 wherein one gene is expressed.

4. A method of claim 1 wherein a first gene and a second gene are expressed.

5. A method of claim 4 wherein said first gene is a reporter gene and said second gene is a gene the expression of which is to be studied in said preselected cell type.

6. A method of claim 4 wherein said first gene is a reporter gene and said second gene is an antisense oligonucleotide.

7. A method of claim 5 or 6 wherein said reporter gene is green fluorescent protein.

8. A method of claim 5 or 6 wherein said second gene is under the control of an inducible promoter.

9. A method of claim 8 wherein said inducible promoter is a heat shock protein promoter.

10. A method of claim 1 wherein said baculovirus is injected at a concentration of at least 10¹⁰ particles/ml.

11. A method of claim 1 wherein said baculovirus is injected at a concentration of about 10¹⁰ to about 10¹¹ particles/ml.

12. A method of claim 1 wherein said baculovirus is injected at a concentration of about 10¹¹ particles/ml.

13. A misexpression assay which comprises systematically expressing a library of genes in specific preselected cells of a zebrafish according to the method of claim 1.

LO

14. A zebrafish expressing at least one gene in specific preselected cells according to a method of claim 1.

15. A method of reducing or eliminating expression of a gene in L5 specific preselected cells of a zebrafish which comprises injecting into the extracellular space around said preselected cells or their developmental precursors in a zebrafish embryo a baculovirus carrying an antisense oligonucleotide which binds to said gene. 20

16. Use of a baculovirus vector for expression of a gene in zebrafish.

17. A baculovirus vector which comprises a reporter gene driven

_*

25 by a ubiquitous promoter and a second expression cassette containing a ubiquitous or inducible promoter, a multiple cloning site and a polyadenylation sequence.

18. A kit for expression of a gene in specific preselected cells 30 of a zebrafish which comprises a baculovirus vector and at least one construct containing a cell type- or tissue-specific promoter, a multiple cloning site and a polyadenylation sequence.

19. A kit according to claim 18 wherein said construct is a 5 transfer plasmid containing a reporter gene under the control of a promoter.

20. A kit according to claim 19 wherein said promoter is selected from the group consisting of the Xenopus EFIα promoter and the polyhedrin promoter.

21. A kit according to claim 18 which further comprises a second construct containing a promoter active in zebrafish, a multiple cloning site, a myc-tag and a polyadenylation sequence.