Patent Application
20090133138
The present invention relates to a method for producing novel transgenic zebrafish.
The present invention also relates to a new gene fragment and novel transgenic zebrafish.
Transgenic ornamental fish is one sector of the fishery business and belong to entertainment industry with global business. For example, transgenic fish expressing green fluorescence by introduction of a GFP gene fused with a fish-specific gene promoter into fertilized eggs, has been generated using zebrafish (Hamada, K. et al., Mol. Marine Biol. Biotech., 1998. 7, 173-180).
Hsiao et al. disclosed a DNA construct flanked at both ends by inverted terminal repeats (ITRs) to increase the efficient expression of transgenic genes in zebrafish. A uniform transgene expression was achieved in the F0 and the following two generations (Hsiao et al., Developmental Dynamics 2001. 220: 323-336). US 2004/0117866 A1 also disclosed a similar gene fragment for producing red fluorescent zebrafish by α-actin promoter.
Although the transgenic green and red fluorescence zebrafishs have been described, method and condition of generating other transgenic fish with other gene fragment (such as red fluorescent protein expressed by β-actin promoter) is different and cannot be easily deduced from the prior art because of the different strategies of genetic construction, gene expression, gene inheritance and uncertainties of the transgenic technique.
U.S. Ser. No. 10/752,687 constructed the pβ-DsRed2-1-ITR gene fragment for producing transgenic medaka (β-actin form medaka). U.S. Ser. No. 11/235,539 used similar gene fragment for producing transgenic cichlid (β-actin form cichlid). However, the expression of the transgene may be influenced by the copy number of the transgenes, and the interactions between the transgene and its flanking genomic DNA as noted on Fraser et al. (Fraser et al. Current Opinion in Cell Biology 1998. 10:361-365). Fraser et al. reported that the site of transgene integration in the host genome will affect the transgene expression, also called the position effect.
The gene targeting is well established in the mouse; however, gene-targeting protocols have not been developed in the rat despite the establishment more than 16 years ago of the first transgenic rats by pronuclear injection (F Kent Hamra et al. PNAS 2002. 99:931-936). Therefore, the results of similar gene fragment expressed in different species are unpredictable and worth studying.
The individual promoters have different abilities to express report gene expression in ES cell and other cell types. (Chung et al. STEM CELLS 2002. 20:139-145) Thus, the same gene driven by promoters from different species is unpredictable of its expression.
The present invention provides a gene fragment comprising (1) a β-actin gene promoter of zebrafish; (2) a gene encodes red fluorescent protein; (3) SV 40 poly-A signal; and (4) inverted terminal repeats (ITR) of adeno-associated virus.
The present invention also provides a method of producing zebrafish with systemic red fluorescence comprising:
The present invention further provides a zebrafish with systemic red fluorescence produced from the set forth method.
The current invention is of thorough and careful design with conceptual breakthrough. A plasmid construct, pZβ-DsRed2-1-ITR, could be generated by introducing the β-actin gene promoter of zebrafish into expression vector pDsRed2-1-ITR (Clontech). The appropriate amount of pZβ-DsRed2-1-ITR is then micro-injected into the cytoplasm of fertilized eggs of zebrafish prior to the first cleavage. These eggs are screened to find progeny expressing fluorescence throughout their systemic tissue. Progeny with fluorescent transgene are then used for future breeding. The term “zebrafish” in the invention is not limited but to that from D. acrostomus, D. aequipinnatus, D. malabaricus, D. albolineatus, D. annandalei, D. apogon, D. apopyris, D. assamensis, D. choprae, D. chrysotaeniatus, D. dangila, D. devario, D. fangfangae, D. frankei, D. fraseri, D. gibber, D. interruptus, D. kakhienensis, D. kyathit, D. laoensis, D. leptos, D. maetaengensis, D. malabaricus, D. naganensis, D. neilgherriensis, D. nigrofasciatus, D. pathirana, D. regina, D. rerio, D. roseus, D. salmonata, D. shanensis, D. spinosus, Brachydanio frankei, Brachydanio rerio albino and Branchydanio sp.
The gene fragment used in the present invention comprising (1) a β-actin gene promoter of zebrafish; (2) a fluorescence gene; (3) inverted terminal repeats (ITR) of adeno-associated virus; and (4) a basic part from pUC.
The red fluorescent gene can be purchased from BD Bioscience Clontech or Evrogen IP (Russia). The red fluorescent gene is DsRed2-1, DsRed2, DsRed2-N1, DsRed2-C1, TagRFP, pTurbo FP635N or pTurboFP635-C. In the embodiment of the invention, pDsRed2-1 is used as the source of the red fluorescent gene. pDsRed2-1 encodes DsRed2, a DsRed variant engineered for faster maturation and lower non-specific aggregation. DsRed2 contains a series of silent base-pair changes that correspond to human codon-usage preferences for high expression in mammalian cells. In mammalian cell cultures when DsRed2 is expressed constitutively, red-emitting cells can be detected by fluorescence microscopy within 24 hours of transfection. Large insoluble aggregates of protein, often observed in bacterial and mammalian cell systems expressing DsRed1, are dramatically reduced in cells expressing DsRed2. The faster-maturing, more soluble red fluorescent protein is also well tolerated by host cells; mammalian cell cultures transfected with DsRed2 show no obvious signs of reduced viability-in those cell lines tested, cells expressing DsRed2 display the same morphology (e.g., adherence, light-refraction) and growth characteristics as non-transfected controls. pDsRed2-1 is a promoterless DsRed2 vector that can be used to monitor transcription from different promoters and promoter/enhancer combinations inserted into the multiple cloning site (MCS).
The fragment of Claim 1, wherein the β-actin gene promoter of zebrafish is SEQ ID NO.:2.
The fragment of Claim 1, wherein the gene encodes red fluorescent protein is SEQ ID NO.:3
A plasmid comprising the gene fragment of Claim 1.
The method of the invention provides five improvements over other methods currently available:
The present invention provides a method of producing transgenic zebrafish with systemic fluorescence comprising:
Accordingly, the preferred linearized construct is selected from
The preferred fluorescent gene used in the method of the invention is red fluorescent gene from pDsRed2-1.
In the method of producing transgenic zebrafish of the invention, the appropriate amount of linearized plasmid construct injected into the fertilized eggs is sufficient to introduce transgene into germ cell of zebrafish. The preferred amount of linearized plasmid construct injected into the fertilized eggs is 1-10 nl. The most preferred amount of linearized plasmid construct injected into the fertilized eggs is 2-3 nl.
The present invention also provides the transgenic zebrafish with systemic fluorescence produced from the method of the invention. The preferred zebrafish has systemic red fluorescence.
The examples below are non-limiting and are merely representative of various aspects and features of the present invention.
Commercially available plasmid construct, pDsRed2-1 (Clontech) was used to generate the expression vector.
The DsRed 2-1 fragment was from plasmid pDsRed2-1. The CMV promoter and two adeno-associated virus inverted terminal repeats (ITR) were ligated to the DsRed2-1 fragment as depicted in
Generating the Novel Plasmid Construct: pZβ-DsRed2-1-ITR
As illustrated in
As illustrated in
As illustrated in
Appropriate amount of pZβ-DsRed2-1-ITR was digested with proportional amount of Not I restriction enzyme. A small fraction of the digested product was analyzed by agarose gel electrophoresis to verify its linearity. The fragment length was 9051 bps as expected.
All DNA plasmids were prepared via ultra-centrifugation with cesium chloride and ethidium bromide gradient (Radloff et al., 1967 Proc Natl Acad Sci USA 57:1514-1521). All DNA fragments used for microinjection were eluted from agarose gel following electrophoresis.
Fish were maintained under artificial conditions of 14 h light and 10 h darkness at 26° C. and maintained on a diet of Tetramin (Tetra, Germany). Before the incubator entered the dark cycle, fish were collected and separated by separation net. On the next morning after the light cycle has begun, fish eggs were collected every 15-20 minutes.
Eggs were collected within 30 minutes of fertilization and attaching filaments removed. The linearized construct was quantified and dissolved in 5×PBS with phenol red at the desired concentration. DNA was picked up by micro-capillary of zebrafish microinjector (Drummond) wherein the injection needle width of the micro-capillary was approximately 10 μm. As micro-needle enters the cell cytoplasm, the DNA injected was approximated 2-3 nl. In each microinjection session, 30-40 eggs were injected; 250-300 eggs were injected in each experiment. Injected eggs were incubated at 26° C. in distilled water.
Injected eggs were rinsed with sterilized solution, cultured in incubator wherein the temperature was 28.5° C. The fluorescence could be observed in the developing embryo after 24 hours.
Embryos were observed under a bright field with a dissecting stereomicroscope (MZAPO, Leica, Germany). Dark field illumination for detecting green fluorescence was performed with a stereomicroscope equipped with a GFP Plus filter (480 nm). The distribution and intensity of the red fluorescence is observed under fluorescence microscope (Leica MZ-12; Fluorescence System: light source Hg 100 W; main emission wavelength 558 nm, and main absorption wavelength 583 nm, filter set RFP-Plus; photography system MPS60). Photographs were taken using an MPS60 camera loaded with ISO 400 film and equipped with a controller for film exposure time (Leica, Germany). In order to examine the distribution of RFP expression in the tissues of transgenic fish, 11 days of post-fertilization larva which having RFP expression on appearance were sectioned and observed under fluorescent microscopy. Larva were fixed for 30 min in 4% paraformaldehyde at 4° C., embedded in cryomatrix (Shandon, USA) and frozen at −20° C. Cryostat sections (Cryostat Microtome, HM500 OM, Microm, Germany) with 15 μm thickness were mounted on slides and observed the RFP fluorescence immediately.
The red fluorescence fish generated from expression vector pp-DsRed2-1-ITR are shown in
As shown in
While the invention has been described and exemplified in sufficient detail for those skilled in this art to produce and use it, various alternatives, modifications, and improvements should be apparent without departing from the spirit and scope of the invention.
One skilled in the art readily appreciates that the present invention is well adapted to carry out the objects and obtain the ends and advantages mentioned, as well as those inherent therein. The cell lines, embryos, animals, and processes and methods for producing them are representative of preferred embodiments, are exemplary, and are not intended as limitations on the scope of the invention. Modifications therein and other uses will occur to those skilled in the art. These modifications are encompassed within the spirit of the invention and are defined by the scope of the Claims.
It will be readily apparent to a person skilled in the art that varying substitutions and modifications may be made to the invention disclosed herein without departing from the scope and spirit of the invention.
All patents and publications mentioned in the specification are indicative of the levels of those of ordinary skill in the art to which the invention pertains. All patents and publications are herein incorporated by reference to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference.
The invention illustratively described herein suitably may be practiced in the absence of any element or elements, limitation or limitations, which are not specifically disclosed herein. The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention that in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention Claimed. Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification and variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention as defined by the appended Claims.
Other embodiments are set forth within the following Claims.
