CN1949979A - Compositions and methods for modifying the content of polyunsaturated fatty acids in biological cells - Google Patents

Abstract

The present invention features compositions (e.g., nucleic acids encoding fat-1, optionally and operably linked to a constitutively active or tissue-specific promoter or other regulatory sequence and pharmaceutically acceptable formulations including that nucleic acid or biologically active variants thereof) and methods that can be used to effectively modify the content of PUFAs in animal cells (i.e., cells other than those of C. elegans, for example, avian or fish cells such as myocytes, neurons (whether of the peripheral or central nervous system), adipocytes, endothelial cells, and cancer cells). The compositions and methods include a fat-1 gene that has been modified to include at least one optimized codon. The modified cells, whether in vivo or ex vivo (e.g., in tissue culture), transgenic animals containing them (fish and birds in particular), and food products obtained from those animals (e.g., meat or other edible parts of the animals (e.g., liver, kidney, or sweetbreads)) are also within the scope of the present invention.

Description

Be used to change the composition and the method for content of polyunsaturated fatty acids in biological cells

Related application

The application's case is advocated in No. the 60/542098th, the U.S. Provisional Application case of on February 4th, 2004 application with in the priority of No. the 60/555422nd, the U.S. Provisional Application case of application on March 22nd, 2004.The full content of two application cases also is included among the present invention as a reference.

Government supports

Part work of the present invention is accepted from national sanitary institute (National Institutes of Health) subsidy (CA79553).Therefore U.S. government has certain right of the present invention.

Technical field that the present invention belongs to

The invention relates to the composition and the method that change content of polyunsaturated fatty acids in the zooblast.

[background technology]

(Polyunsaturated Fatty Acids PUFA) is meant that carbon number is the fatty acid that contains two or more pairs key in 18 or more a plurality of and the carbochain to polyunsaturated fatty acids.The position of double bond of the most close fatty acid methyl end of foundation they can be divided into two big class: n-6 and n-3 (please refer to Gilland Valivety, Trends Biotechnol. 15: 401-409,1997; Broun et al., Annu.Rev.Nutr. 19: 197-216,1999; Napieret al., Curr.Opin.Plant Biol. 2: 123-127,1999).The synthetic of n-6PUFA and n-3 PUFA is to pass through respectively linolenic acid (Linoleic Acid, LA, 18:2n-6) and α-linolenic acid (α-Linolenic Acid, ALA, 18:3n-3) carry out a series of dehydrogenations and prolongation and finish (Gill and Valivety, as described above; Broun et al., as described above; Napier et al., Napier et al.).One of main end product of n-6 synthesis path is arachidonic acid (ArachidonicAcid in the animal body, AA, 20:4n-6), the main end product of n-3 synthesis path is arachic acid (Eicosapentaenoic Acid, EPA, 20:5n-3) and DHA (Docosahexaenoic Acid, DHA, 22:6n-3).

Participating in the synthetic important enzyme material of a wherein class of PUFA is a class fatty acid dehydrogenase.This fermentoid is introduced two keys according to the position of the specificity of enzyme decision in carbochain.Though under most of situation, comprised the enzymic activity that LA (18:2n-6) and ALA (18:3n-3) is converted into the PUFA (wherein conversion ratio is restricted) of long-chain in the animal body, but they lack synthetic precursor (precursor) PUFA, the necessary 12-of LA, ALA and the 15-dehydrogenase activity (please refer to Knutzon et al., J.Biol.Chem. 273: 29360-29366,1998).And, n-3 and n-6 PUFA in mammalian cell be can not transform (please refer to Goodnight et al., Blood 58: 880-885,1981).Therefore, prolongation product, the dehydrogenation product of LA and ALA and their correspondences thereof are considered to the essential fatty acid of taking in the human foods.To a great extent, the PUFA composition of mammalian cell membrane is to rely on the absorption of food (to please refer to Clandinin et al., Can.J.Physiol.Pharmacol. 63: 546-556,1985; McLennan et al., Am.Heart J. 116: 709-717,1988).

On the contrary, certain plants and microorganism contain membrane-binding 12-and 15-(n-3) dehydrase, this dehydrase can be subjected to matter generation effect with glyceride in plasmid and endoplasmic reticulum, thereby these plants and microorganism can be synthesized such as the n-3 fatty acid of ALA (18:3n-3) and (be please refer to Browse andSomerville, Annu.Rev.Plant Physiol.Plant Mol.Biol. 42: 467-506,1991).Gene technology has realized that the gene that picks out coding 12-and 15-dehydrase from arabidopsis (Arabidopsis thaliana) and other higher plant kind (please refer to Okuley et al., PlantCell 6: 147-158,1994; Arondel et al., Science 258: 1353-1355,1992).Recently, a kind of fat-1 gene of the n-3 of coding fatty acid dehydrogenase selects from Caenorhabditis elegans (nematode) and grows (clone) and come out (to please refer to Spychalla et al., Proc.Natl.Acad.Sci.USA 94: 1142-1147,1997; U.S. Pat 6194167).

Summary of the invention

The present invention be the part based on above-mentioned C.elegans n-3 dehydrogenase gene fat-1 can successfully be incorporated into other type zooblast (as the cell of mammal, birds and fish) thus in improve n-3 PUFA content and balance n-6 in the cell fast and effectively: in this discovery of the ratio of n-3 PUFA.We have also found modification C.elegans fat-1 nucleotide sequence.When the codon that self forms that changes one or more encoding amino acid sequence during same acid sequence, is " optimization " sequence with this sequence definition but this codon is still encoded.For example, can replace by CTG with one or more codon of " GTT " expression, coding valine (valine), and this CTG valine of encoding equally; One or more codon with " CGT " expression, coding arginine (arginine) can be replaced by CGC, and this CGC arginine of encoding equally; Or the like.Codon after the modification makes the easier insertion recombinant DNA of organism.Thus, comprise be encoded to have with the dehydrogenation of omega-6 fatty acid be omega-3 fatty acid enzymic activity albumen sequence nucleic acid (this nucleic acid comprises from original or self generation state nucleic acid through codon optimized modification) and use those sequences to produce transgenic animal (as mammal, birds or fish) all within the scope of the invention in order to do the method that makes treatment or help all kinds of diseases of prevention or illness.Can be introduced in into following any carrier and the cell through optimized sequence, and any method of wild-type sequence (as the fat-1 gene) of using is also applicable to this optimization.(in the production as transgenic animal) in some cases is more suitable for adopting codon optimized sequence.Optimization is not the unique method of unmodified nucleic acid sequence.The present invention includes to use and be encoded to omega-3 (or n-3) dehydrase or optimize the bioactive fragment of omega-3 (or n-3) dehydrase or the sequence of other mutant.Enzymic activity activated protein or enzyme with omega-3 dehydrogenase activity are the albumen that n-6 PUFA is converted into n-3 PUFA in cell interior.Albumen that those albumen or enzyme may form with their corresponding self is by identical length and form (as the albumen for coding fat-1), perhaps they may be fragment or other mutant of this albumen or enzyme, and it has the enough enzymic activitys that can be used for one or more method of the present invention.

Know clearly it, discover, the performance of the xenogenesis of fat-1 gene makes those cells various n-6 PUFA can be converted into corresponding n-3 PUFA in the mouse myocardium cell, thereby the ratio of n-6: n-3 is changed into about 1: 1 (desired proportion) from 15: 1 (not desired proportion).Using " xenogenesis performance " this word is to be used for showing that the intracellular sequence of an appointment is not the sequence of the normal performance of this cell; This sequence is because be the sequence of different plant species and be called as " xenogenesis ".In addition also find, obviously descend from the content of arachic acid (as arachidonic acid) in transgenic cell of n-6 PUFA.Can further tell about hereinafter, can judge whether given nucleic acid is encoded and had the enzyme of omega-3 dehydrogenase activity by measuring arachidonic contents level; Similarly, also can pass through to measure the contents level of n-6 PUFA, contents level and/or the n-6 of n-3 PUFA: the ratio of n-3 PUFA.Thus, the invention is characterized in the composition and the method that can be used for effectively changing PUFA content in the zooblast, said composition promptly be encoded to have the omega-3 dehydrogenase activity polypeptide (for example, fat-1), it is optionally and be operably connected to continuous activation or tissue-specific promotor.This cell can be the cell of mammal, birds or fish, as long as can also be any cell that comprises n-6 PUFA.For example, this cell can for myocyte, nerve cell (in around or among the central nervous system), adipocyte, endothelial cell, liver cell or cancer cell (comprising colon, mammary gland, liver, prostate, ovary and Cervical cancer cell).Following experiment mainly is the sequence at C.elegans fat-1, yet the present invention is not limited to this; Composition of the present invention and method comprise those nucleic acid that comprise (or use) any coding and have the polypeptide of omega-3 dehydrogenase activity (for example, no matter be self whether form, no matter be that whether any of C.elegans can be the polypeptide of corresponding omega-3 fatty acid with the dehydrogenation of omega-6 fatty acid).Thus, in specific embodiment, method of the present invention is so that n-6 fatty acid is converted into n-3 fatty acid in animal body is feature by producing n-3 fatty acid dehydrogenase (being the omega-3 dehydrogenase activity) in animal body.Can when in animal body, throwing the nucleic acid that gives coding n-3 fatty acid dehydrogenase or its biologically active variant, begin to produce this n-3 fatty acid dehydrogenase.The present invention will provide the specific embodiment of this nucleic acid, and those know those skilled in the art scholar especially is easy to discern similar functions under the guidance that the present invention provides subsequently sequence.Any animal species of mentioning among the present invention includes at interior (as any species in mammal, birds or the fish), and these animals itself may have average or " normally " ability that produces the n-3 fatty acid dehydrogenase, also may not have.For example, method of the present invention can apply in animal (as the mankind) body of before the dehydrase throwing is given (undesirably) limited in one's ability of generation n-3 fatty acid.

Nucleic acid (as fat-1 sequence or its biologically active variant) can be connected to the adjusting sequence by feasible pattern.This adjusting sequence not only comprises promotor, comprises that also enhancer or other are beneficial in the cell inner expression expression of nucleic acids control sequence through modification, as the polyadenylic acid signal.This cell through modification can be placed in the body, also can be retained in external (as in the tissue culture ware).Those cells that from their self environment, shifted out all be " through single from ", and when take nucleotide sequence as described herein (nucleic acid in the heterogenous cell may with described " through singly from " nucleic acid is similar) time includes within the scope of the invention.The present invention also comprises and has described nucleic acid or through non-human, the transgenic animal of the cell of modification, and from the food (as meat or other edible part (as liver, kidney, skin, fat or pancreas) of animal) of these animals or with the food of animal body position processing (as meat soup, gravy, sauce or any by transgenic animal position (as the position of ox or chicken) making through processed food).These foods can provide to the mankind and consume, and also can be the food of feeding pet or domestic animal.

It should be noted that when further transgenic animal being discussed that these transgenic animal (or tissue of any appointment) may be also may not be self just to express the omega-3 dehydrase is arranged; Present invention resides in self and lack the method that by the transgenosis mode n-6 fatty acid is converted into n-3 fatty acid in the animal body that n-3 fatty acid dehydrogenase ability is provided.Described method and composition also can be used in animal body (as mammal, birds or fish) and improves or additional n-3 fatty acid dehydrogenase activity.In one embodiment, the invention is characterized in that containing nucleotide sequence (may pass through and optimize, may not have yet) the cells of mammal, birds or fish, wherein this nucleotide sequence is to compile n-3 dehydrase (as C.elegant n-3 dehydrase) or its biologically active variant (as fragment or other mutant), and this variant comprises the variant (variant as shown in Figure 18) of coding C.elegant fat-1 albumen.Can see above, the biologically active variant of n-3 dehydrase be meant those n-3 dehydrases that kept wild type treatment or clinically effectively enough bioactive variant (promptly the treatment patient, produce transgenic animal, diagnose or other experiment test in useful variant).For example, the variant of n-3 dehydrase can be to have kept the mutant or the fragment of the bioactive enzyme of the wild type n-3 dehydrase of 5-25% at least.Such as, the fragment of n-3 dehydrase is the biologically active variant fragment of the enzyme of total length, the conversion ratio that this fragment changes into n-3 fatty acid with n-6 fatty acid be at least mutually deserved wild-type enzyme under the same case 5-25% (as conversion ratio be wild type n-3 dehydrase 5,10,20,30,40,50,75,80,90,95 or 99%).The conversion situation that n-6 fatty acid is converted into n-3 fatty acid also can be used for helping to assess by the codon optimized modification sequence that obtains (can be used for judging that as this conversion situation the biologically active of those sequences obtains having kept or being improved).

Variant also may comprise one or more amino acid whose replacement, delete or increase (as in the wild-type enzyme sequence 1%, 5%, 10%, 20%, 25% or more amino acid residue can be replaced by another amino acid residue or delete).Describedly different be that the nucleic acid variant in the protection domain of the present invention can have maybe can comprise the sequence consistent with wild type n-3 dehydrogenase gene (as the fat-1 of C.elegans) at least 75%, 80%, 90%, 95% or 99%.Similarly, the nucleic acid variant codified in the protection domain of the present invention has the albumen of the sequence consistent with wild type n-3 dehydrase (as the Fat-1 of C.elegans) at least 75%, 80%, 90%, 95% or 99%.Wherein this variant comprises the substituent that can form the amino acid retention displacement that this technical field knows.The nucleotide sequence that changes to described degree from wild type needs only the albumen of retains biological activity and/or coding biologically active promptly applicable to method of the present invention.

The convenient system that is provided for characterizing the functional character of fat-1 gene and product thereof owing to the cell (optionally being operably connected to continuous activation or tissue-specific promotor and/or other adjusting part) of expressing the fat-1 sequence seems especially valuable.Cell in tissue culture is especially convenient, but the present invention is not limited to this.Do not have the required lengthy process of present cultured cell or animal, can study the celelular mechanism that any n-3 of having fatty acid participates in yet through the Fat-1 of modification cell.This cell also can be used as model system, and for example, this model system is to be used for assessing existing method and to design new method for the sequence transfer of the n-3 dehydrase of will encode effectively in vivo enters cell.

(no matter composition (as nucleic acid structure) is to be used for the treatment of patient as described herein in any described content, still produce transgenic animal, still in cell culture assays), coding have omega-3 dehydrogenase activity (as fat-1) polypeptide nucleic acid can with another (or " second ") gene co expression (by same vehicle or use the mode of the carrier separately of identical or different type).For example, second gene (looking fat-1 is first gene) can be another therapeutic gene (as the acceptor of little molecule or chemotherapeutant) or indicate gene (as the sequence of coding fluorescence albumen, as green fluorescent protein (GreenFluorescent Protein, GFP) or enhanced GFP (EGFP)).The variant fat-1 nucleic acid that comprises the fat-1 nucleic acid through optimizing may reveal the albumen that is better than its corresponding wild-type by coding schedule.For example, this variant sequence may be more effective when expressing dehydrase (in tissue culture or cell in vivo).

Any nucleic acid molecules of the present invention can be " through single from " (as residing environment be different from their abiogenous environment (see also when describing codon optimized sequence relevant use determiner " through single from " tell about)).For example, this nucleic acid can be added in plasmid or other carrier, or is connected on one or more xenogenesis sequence, comprises the adjusting part such as promotor and enhancer.When nucleic acid is contained in the heterogenous cell (as not by the cell of normal expression), this nucleic acid molecules also through single from.For example; in mammalian cell (as the cell of human, ox or pig), birds cell (as the cell of chicken, duck or goose) or the fish cell (as the cell of salmon, trout or yaito tuna), the nucleic acid (or its variant) that includes the C.elegansfat-1 gene be through single from nucleic acid.The cell of the described fat-1 of having all is in protection scope of the present invention.

Nucleic acid of the present invention also can be formulated into to be used for throwing and gives patient.For example, they can be in sterile water or in the sterile physiological buffer (as the phosphoric acid buffer saline solution) with oral or parenteral take (as injection, mucous membrane in intravenous injection, intramuscular injection, the corium penetrate injection, corium penetrates injection or hypodermic injection) throwing gives patient.This composite also can be prepared into and be applied to tissue or organ (as with solution, gel or paste mode).In the patient body, have in the situation of tumour, composition of the present invention can be expelled in the tumour or throw and give tumour and cut off in the tissue around the position.

Feature of the present invention also is to express the transgenic animal (comprise any domestic animal or as food source (as fish or other aquatic animal (as squid, octopus, shellfish (as lobster, crab, snail and shrimp) or other edible aquatic animal (eel)))) of C.elegans n-3 dehydrase or its biologically active variant.When sending into C.elegans fat-1 gene in the zooblast, this gene can be expressed this discovery effectively, and this gene and variant thereof and other fat-1 gene can produce transgenosis mouse or bigger transgenic animal (as ox, pig, sheep, goat, rabbit or other domestic animal or domestic animal according to method well known in the art; Any edible birds (as chicken, turkey, goose, duck or pheasant); And the fish that comprise shell aquatic organism and shellfish).More detailed it, the present invention includes to be used for producing and comprise cod (or any gadidae, Gadiformes fish (as haddock)), halibut (common names of two kinds of flat fishes of mediocre butterfly fish), catfish (the common name of several fish in the Pacific herring order fish, wherein also comprise long tail anchovy), mackerel (common name of various import edible fishes in the mackerel section fish), salmon (or any salmon fishes, comprise trout), perch (or any bacalao), ray (or any Rajidae fish), flatfish (or any Osmeridae fish), sole (or any Soleidae fish), and yaito tuna (or any mackerel section fish) (wherein transgenosis is the composition and the method for omega-3 dehydrase or its biologically active variant (as the C.elegansfat-1 gene, it can comprise at least one optimizing codon) interior transgenosis fish.

Whether comprise the promotor or the tissue-specific promotor of tool of continuous activation according to used molecular configuration, the expression way of omega-3 dehydrogenase gene (as the fat-1 gene) can be popularity or tissue specificity mode.When placing control following time of the promotor of continuous activation, can express gene in many types of organizations, can express (the promotor that for example has selective active in particular organization or cell type when the control following time that places tissue-specific promotor in particular cell types, this particular cell types comprises skeletal muscle tissue, cardiac muscular tissue, smooth muscle tissue, breast tissue, colon, the cell of prostata tissue, neuron, the retina cell, pancreatic cell (as islet cells), other endocrine cell, endothelial cell, Skin Cell, adipocyte, or the like).

As described later, the cell of transgenic animal can comprise the PUFA content through changing, and this PUFA is beneficial to edible more.Like this, breeding transgenic livestock (or expression is by any edible animal (as fish or pheasant) of the dehydrase of fat-1 gene code) will be the food source of more excellent (as more healthy).The food that obtains from these animals or the food product (as food and the pet food through processing) that uses these animal processings can be supplied with healthy individual or supply suffers from one or more disorder described herein.

Be noted that, feature of the present invention also is that treatment suffered from and maybe may develop and n-3 PUFA deficiency or n-3: the patient's of n-6 PUFA relevant illness out of proportion method, this method is the nucleic acid that gives coding n-3 dehydrase or its biologically active variant (as fragment, mutant or codon optimized sequence) by throwing.Also can throw and give by nucleic acid or biologically active variant encoded protein.This methods of treatment can be used for cardiac arrhythmia or angiocardiopathy (for example high fat of blood or hypertension) patient, cancer (as breast cancer or colon cancer) patient, inflammation or autoimmune disease are (as rheumatoid arthritis, multiple sclerosis, enteritis (IBD), asthma, COPD, lupus, diabetes, the Si Yegelun syndrome (Sjogren ' s syndrome), the anchylosis rachitis, polyarteritis nodosa, the Lai Teer syndrome (reiter ' ssyndrome), or chorionitis) patient, or retina or brain deformity (or occur among the premature tendency sex deviation) patient.The patient who is fit to also comprise those suffered from or diagnose diabetes are arranged, the patient of obesity, cutaneous disorder, ephrosis, ulcerative colitis or Crohn's disease (Crohn ' s disease).Other patient who is suitable for comprises that those are in the high-risk patient of organ transplant rejection.The expression of fat-1 can suppress cell death (when cell death takes place by Apoptosis (apoptosis)) in such as neuronal cell, method of the present invention so also can be used for treatment or prevention neurodegenerative disease (for example, suppressing possibility or duration or the seriousness that this disease takes place).Thus, the invention is characterized in treatment patient's method, this patient has suffered from or has been about to develop into such as Parkinson's disease (Parkinson ' s disease), Alzheimer's (Alzheimer ' s disease), Huntington (Huntington ' s disease, HD), spinal and bulbar muscular atrophy (spinal and bulbar muscular atrophy, SBMA, also claim the Ken Nidishi disease), dentate nucleus rubrum globus pallidus Louis body atrophy (dentatorubral-pallidoluysian atrophy, DRPLA), spinocerebellar ataxia the 1st type (SCA1), SCA2, SCA6, SCA7 or horse are looked into many-Joseph disease, and (Machado-Joseph disease MJD/SCA3) (please refer to Reddy et al.Trends Neurosc. 22: 248-255,1999).

The expression of fat-1 also can be promoted the cell death of cancer cell, so the method for the present invention's sign can be used for treating or prevention (aggravating or diffusion as inhibition generation, inhibition) cancer cell, comprising breast cancer, colon cancer, prostate cancer, liver cancer, cervix cancer, lung cancer, the cancer of the brain, cutaneum carcinoma, cancer of the stomach, Head and Neck cancer, cancer of pancreas, leukemia (as leukemia and lymph cancer) and oophoroma.Especially, this method can adopt and have the sequence that the nucleic acid of optimizing C.elegans fat-1 gene is constructed body (construct) and optionally had second albumen (as chemotherapy albumen) of encoding.This is constructed body and also can be adjusting sequence (as continuous activation or cell type-specific promotor).Be used for the treatment of or the nucleic acid of prophylaxis of cancer can be formulated into to be used for throwing and gives to patient.For example, nucleic acid is mixed in aseptic, the physiologically acceptable dilution.If tumour grows up to, nucleic acid can be formulated into injection solution.

The balanced ratio of n-6: n-3 or better balanced ratio are for normal development and to grow up be very important, and as noted earlier, method of the present invention applies to those and suffers from the unrecognizable disease relevant with PUFA or the patient of illness has superiority on one's body.

Comprise herein the following title of writing a Chinese character in simplified form: AA represent arachidonic acid (Arachidonic Acid, 20:4n-6); DHA represent DHA (Docosahexaenoic Acid, 22:6n-3); EPA represent arachic acid (Eicosapentaenoic Acid, 20:5n-3); GFP represents green fluorescent protein (Green Fluorescent Protein); Ad.GFP represents to have the adenovirus of GFP gene; Ad.GFP.fat-1 represents to have simultaneously the adenovirus of fat-1 gene and GFP gene; And PUFA represents polyunsaturated fatty acids (Polyunsaturated Fatty Acid).

Similar or be equal to method as herein described and material can the present invention actual use and test in use, but the present invention also can disclose other useful method and material below.For making any United States Patent (USP) of issuing from the application's case smooth, all references that all public publication, patent application document, patent document and other this paper quoted all comprise in the present invention as a reference.

Other features and advantages of the present invention will disclose in below detailed description, accompanying drawing and the specific embodiment one by one.

Description of drawings

Fig. 1 is four microphotos that show transgene efficiency.Represent that respectively rat myocardial cell infects Ad.GFP (one hurdle, the left side; Control group) or Ad.GFP.fat-1 (one hurdle, the right).Infect after 48 hours, use light (going up the hurdle) and 510nm blue light (following hurdle) irradiation back visualize cardiac muscle cell respectively.The coexpression of GFP it seems that from range estimation transgenosis can come out with high-efficient expression cell.

Fig. 2 is one group of autoradiograph, in order to be illustrated in ribalgilase (RNase) protection test of fat-1 transcriptional level in the cardiac muscle cell who infects Ad.GFP (control group) respectively and infect Ad.GFP.fat-1.From the cardiac muscle cell, extract total RNA (10 microgram) back and antisense RNA probes hybridization, shear and polyacrylamide gel electrophoresis separation by sex change through RNase.The messenger mrna of fat-1 can be by the autoradiograph video picture.The probe that points to β-actin gene is as control group.

Fig. 3 is the analysis of spectra of a pair of part gas chromatography, in order to represent self-infection respectively Ad.GFP with reference to the cardiac muscle cell and infected the fatty acid spectrogram of total cytolipin that the cardiac muscle cell of Ad.GFP.fat-1 extracts.

Fig. 4 describes respectively at the PGE of control group cardiac muscle cell with the cardiac muscle cell who expresses the fat-1 gene ₂The bar chart of level (by the enzyme immunoassay (EIA) decision).Numerical value is the mean value ± SD of three tests among the figure, and is expressed as control group %. ^*p＜0.01。

Fig. 5 represents respectively from the control group cardiac muscle cell and expresses the form that polyunsaturated fatty acids is formed in total cytolipin of transgenosis cardiac muscle cell of C.elegans fat-1 cDNA.

Fig. 6 is the flow chart of experimental arrangement.

Fig. 7 is the flow chart of experimental arrangement.

Fig. 8 is the flow chart of experimental arrangement.

Fig. 9 is the analysis of spectra of a pair of part gas chromatography, in order to represent respectively from the fatty acid spectrogram of control group neuron with total cytolipin of the neuron extraction of having infected Ad-GFP-fat-1.

Figure 10 is comparison from rat cortical neuron (control group) and the form formed from polyunsaturated fatty acids in total cytolipin of the transgenic cell of expressing C.elegans fat-1 cDNA (fat-1).

Figure 11 describes respectively at control group neuron and the neuronic PGE of expressing the fat-1 gene ₂The bar chart of level.Neuronic PGE through the Ad-GFP-fat-1 infection ₂Level is lower than control group neuron.Numerical value is the mean value ± SD of three tests among the figure, and is expressed as control group %. ^*P＜0.01。

Figure 12 represents the MTT analysis result bar chart of cell survival rate in the culture of control group and expression fat-1 respectively.Removing growth factor-2 after 4 hours, the cell survival rate of the neuronal cell of expression fat-1 gene is than cellular control unit high by 50% (p＜0.01).

Figure 13 is the tracking map of the differential responses of a pair of expression outer 7.5mM calcium of myocyte's pair cell that infected by Ad.GFP and infected by Ad.GFP.fat-1.

Figure 14 is that (0-4 week behind the injecting virus) gross tumor volume changes line chart after expression a period of time, and gene transfer that hence one can see that is to the influence of tumor growth.Breast cancer cell (MDA-MB-231) is with the subcutaneous nude mice back that is implanted to.After three weeks, by the intratumor injection mode to mouse respectively with Ad.GFP.fat-1 or Ad.GFP (control group; 50 μ l, 10 ¹²VP/m) treatment.

Figure 15 shows from control group MCF-7 cell and the form formed from polyunsaturated fatty acids in total cytolipin of the transgenosis MCF-7 cell of expressing C.elegans fat-1 cDNA.

Figure 16 describes respectively at the PGE of control group MCF-7 cell with the MCF-7 cell of expressing the fat-1 gene ₂The enzyme immunoassay (EIA) of level is bar chart as a result.Numerical value is the mean value ± SE of three tests among the figure, and is expressed as control group %.( ^*P＜0.05)。

Figure 17 A and 17B represent the nucleotide sequence of C.elegans fat-1 cDNA and the Fat-1 amino acid sequence of polypeptide of deriving respectively.

Figure 18 is that the fat-1 nucleotide sequence is optimized in expression.

Figure 19 is the analysis of spectra of a pair of part gas chromatography, in order to expression respectively from the different polyunsaturated fatty acids spectrograms of the TL of the skeletal muscle of wild type mouse (WT, last hurdle) and fat-1 transgenosis mouse (TG, following hurdle) extraction.Wild type mouse and transgenosis mouse all are big female mouse of 8 weeks, and the food of being fed is the same.The level of n-6 polyunsaturated fatty acids (18:2n-6,20:4n-6,22:4n-6 and 22:5n-6) is all very low in the transgenosis muscle (following hurdle), yet n-3 fatty acid (is used ^*Mark) many than wild type muscle (go up hurdle).In the wild type animal body, the polyunsaturated fatty acids in the tissue main (98%) be the n-6 linolenic acid (LA, 18:2n-6) and arachidonic acid (AA 20:4n-6) has (or can't be detected) n-3 fatty acid on a small quantity.On the contrary, a large amount of n-3 fatty acid is arranged, comprising linolenic acid (ALA in the tissue of trangenic mice, 18:3n-3), arachic acid (EPA, 20:5n-3), clupanodonic acid (DPA, 22:5n-3), DHA (DHA, 22:6n-3).Thus, the level of n-6 fatty acid LA and AA significantly reduces in genetically modified organism, shows that n-6 transforms to n-3 fatty acid.In the transgenic animal tissue, the final ratio of n-6 and n-3 fatty acid is near 1.Listed as table 1, can see that in the organ-/ tissue of all tests the lipid that is rich in n-3 has n-6: the balanced proportions more of the balanced proportions of n-3 and AA/ (EPA+DPA+DHA).

Figure 20 is the analysis of spectra of a pair of part gas chromatography, in order to expression respectively from the different polyunsaturated fatty acids spectrograms of wild type zebra fish with the TL that extracts through the muscular tissue of the transgenic zebrafish of the expression fat-1 of embodiment 8 described modifications gene.

Figure 21 is the analysis of spectra of a pair of part gas chromatography, in order to expression respectively from wild type pig and different polyunsaturated fatty acids spectrograms through the TL of the afterbody tissue extraction of the transgene pig of the expression fat-1 of embodiment 8 described modifications gene.

Summary of the invention

Following studies show that, but be not limited only to this, the nucleic acid molecules of coding n-3 dehydrogenase can be in various zooblast forms effective expression out, the result, those cells produce a large amount of n-3 PUFA from endogenous n-6 PUFA, thereby have the n-6 than balance: n-3 PUFA ratio (1: 1). What use when carrying out this research is can be in vivo or the recombinant adenoviral expressing vector of external medium transgenosis. The adenovirus vector of expression omega-3 dehydrogenase (such as fat-1) or its biologically active variant (such as codon optimized sequence) and the virus of other form and non-viral expression vector are all in protection scope of the present invention.

In details of the words, the present invention is converted into n-6 the nucleic acid of sequence of dehydrogenase of corresponding n-3 aliphatic acid as feature to comprise coding. Although our research concentrates on the dehydrogenase through C.elegans fat-1 gene code, the sequence of other dehydrogenase of encoding also can be included in nucleic acid of the present invention and construct in the body and be used in the method for the present invention. For example, encoded dehydrogenase can be plant, the nematode except C.elegans, blue-green alge or the fungi (such as different branch water mold (Saprolegnia diclina)) of being rich in EPA. Other can provide the fungi of dehydrogenase sequence to comprise saccharomycete (Saccharomyces kluyveri) and different branch water mold. Therefore, nucleic acid molecules of the present invention comprises the sequence of coding n-3 dehydrogenase, and this sequence is operably connected to (such as continuous activation or tissue-specific promoter) on the adjusting part. Concrete promoter is also can being described in further detail below of knowing in this area.

The sequence of coding n-3 dehydrogenase can comprise at least one optimization codon. The codon quantity of optimizing can be different. Preferably, this quantity is enough to improve the expression (as the tricks of transcribing) of certain aspect or otherwise increases the purposes of this sequence. In some cases, only modification minority codon (such as 1-5) just can strengthen this sequence. In the other situation, need to optimize a large amount of codons (for example, at least 5 and maximum 150). In having embodiment, the initial source of ignoring desaturase coding sequence, nucleic acid molecules can comprise 5-10,10-15,15-20,20-25,25-30,30-35,35-40,40-45,45-50,50-55,55-60,60-65,65-70,70-75,75-80,80-85,85-90,90-95,95-100,100-105,105-110,110-115,115-120,120-125,125-130,130-135,135-140,140-145,145-150,10-125,25-100,30-90,40-80,50-70 or general 60 optimization codons. In an implementation aspect, this nucleic acid molecules can comprise nucleotide sequence shown in Figure 180.

And this position of optimizing codon also can be different. C.elegan fat-1，6、9、18、20、22、 24、28-30、33-36、47、49、52、54、58、60、61、64、 67、69-71、73、77、79、81、86、89、92、94-95、100、 101、105、106、112、115、118、124、127、128、131、 146、151、154、161、163、164、169、178、187、188、 195、197、200、202、206、210、214、217、221、223、 225、227、228、232、234、241、245、255、271、280-282、 284、285、301、303、310、312、327、362370 。 When the gene of the dehydrogenase coding that uses was not C.elegan fat-1 gene, the position of optimizing codon also can be in these positions on one or more (comprising all) position. In gene of the same race (such as the n-3 dehydrogenase gene of plant or fungi), the optimization position can be and corresponding position listed above.

As mentioned above, nucleic acid molecules more of the present invention can be considered to " through single from ". Yet when this nucleic acid molecules was not spontaneous sequence, this restriction was always unnecessary. Owing to unlikely produce the sequence of optimised (especially several codons are optimised) in the nature, thus do not need these sequences are thought " through single from ". Like this, when nucleic acid molecules is spontaneous sequence must be " through single from " (single from the natural environment of its coexistence some other, most of or whole composition), and when the sequence of nucleic acid molecules be not nature do not need to be defined as when producing (as the nucleic acid with non-natural optimization) through list from.

Except adjusting part and desaturase coding sequence, nucleic acid molecules also comprises coding can improve the purposes of this molecule to offer medicine individual useful polypeptide (such as therapeutical peptide) or coding in experimental identification polypeptide (can the markd albumen of coding-belt such as the second sequence). The example of fluorescence labeling (such as GFP and EGFP) and the example of non-fluorescent label (such as beta galactosidase) are provided herein. Other well known and customary mark of using or " report (reporter) " albumen are included in equally described nucleic acid and construct in the body.

Nucleic acid molecules can for or can partly be a carrier (such as expression vector). We use adenovirus vector above-mentioned (but also reference example) in the experiment. Can be used as among the present invention express other viral vectors construct body also comprise from such as vaccinia virus (such as poxvirus or change surname vaccinia virus Ankara (MVA)), with the carrier of adeno-associated virus (AAV) or herpesviral. These viruses often are with when being used for the purposes relevant with zooblast (comprising the human cell) and are served compelling feature. For example, select herpes simplex virus (such as HSV-1) that dehydrogenase (such as fat-1 or its homologue (or biologically active variant, comprise codon optimized variant)) is passed to neuronal cell. This class carrier is of great use in treatment or prevention neurodegenerative disorders.

Retroviruse, liposome and plasmid vector are also well known in the art and can be used for coded sequence with the n-3 dehydrogenase and pass to cell and (can be used for merging desaturase coding sequence (such as fat-1) such as expression vector pUR278 and advance the lacZ gene; The lacZ discernible mark beta galactosidase of encoding (please refer to Ruther et al., EMBO J.2：1791，1983))。

Should be noted, desaturase coding sequence (such as the fat-1 sequence) or its biologically active variant (comprising codon optimized sequence) also can be fused in the xenogenesis sequence of other type, and for example this another therapeutic genes of xenogenesis sequential coding maybe increases the quantity of this fusion or improves the quality (such as solubility or circulating half-life) of this fusion when being expressed. For example, the pGEX carrier can be used for expressing the albumen of the present invention that is fused to glutathione S-transferase (GST). Usually, this fusion is soluble, and can by be adsorbed onto first on glutathione-agarose beads, then in the presence of not having glutathione elution and easily from the dissolving cell in purifying. This pGEX carrier is (from Pharmacia Biotech Inc; Please refer to Smith and Johnson, Gene67: 31-40,1988) be designed to the division position with plasmase or Xa factor protease, in order to do the target gene product through choosing is grown can be spun off from the GST part. Other fusion partners comprises albumin and immunoglobulin molecules (such as IgG, IgA, IgM or IgE) zone (such as the Fc section, having or do not have hinge area). Other useful carrier comprises pMAL (from New England Biolabs, Beverly, MA) and pRIT5 (from Pharmacia, Piscataway, NJ), and both are fused to the n-3 dehydrogenase with maltose E in conjunction with albumen and albumin A respectively.

Transgene expression can be enough to obtain in episome system (episomal system) prolong, and gives transgenosis in order to do making not need any given expression vector thrown again. Perhaps, carrier can be designed to promote to insert the host chromosome group, is preferably in the location specific place, is not lost in the phase at cell survival to guarantee transgenosis. Transmit in which way, by performed the transcribing control and all will promote the specificity of tissue and regulate genetically modified expression of host cell. Thus, nucleic acid molecules of the present invention can comprise the sequence that promotes in the desaturase coding sequence insertion host chromosome group.

Selecting or designing of expression vector is to depend on for example to want to turn the host cell type of shape and the aspiration level of protein expression. For example, when host cell was mammalian cell, expression vector can comprise viral adjusting part, such as the promoter from polyomavirus, adenovirus 2, macrophage virus and simian virus 40 (SV40). These adjusting parts also can be used for nonmammalian (such as birds or fish) cell. The nucleic acid that inserts is (such as the sequence of wish expression; Here adopt such as the n-3 dehydrogenase through the fat-1 coding) also can be modified becomes the sequence of coding residue, and this residue preferably (please refer to Wada et al., Nucleic Acids Res. at E.coli20: 2111-2118,1992) middle use. Equally, if need or expectation, also can be in the organism except E.coli the modification codon. These modifications (as comprising various adjusting parts and codon optimized modification) can reach by the standard recombinant technique. More extensive, expression vector of the present invention can be designed to the carrier of expressing protein in prokaryote or eukaryotic cells. For example, polypeptide of the present invention can be expressed in bacterial cell (such as E.coli), fungi, saccharomycete or insect cell (as using baculoviral (baculovirus) expression vector). Wherein, the baculoviral that for example is grown in the autographa california nuclear polyhedrosis virus (AcNPV) in greedy noctuid (Spodoptera frugiperda) cell in meadow can be used as carrier and express the n-3 dehydrogenase. Yet the present invention is not limited to this, and when expressed main target was the generation of omega-3 dehydrogenase and purifying, we wished that E.coli, saccharomycete and insect cell all can be used as host cell. It should be noted that the present invention also is included in the expression of omega-3 dehydrogenase in the higher mammal cell, comprise that those are in the various multi-form interior expression of transgenic animals.

As mentioned above, when host cell obtains or host cell when being exactly cell in the multicellular animals body from multicellular animals, the nucleic acid that expression vector and being used for is expressed dehydrogenase (such as the fat-1 nucleotide sequence) also can comprise tissue-specific promoter. This promoter is known by the personage of this area, and it comprises that (but being not limited only to this) liver specificity promoter is (such as albumin; Please refer to Miyatake et al., J.Virol.71: 5124-5132,1997), the muscle specific promoter ((please refer to Shi et al., Hum.Gene Ther. such as myosin light chain 18: 403-410,1997) or α-actin actin), pancreas specificity promoter (such as insulin, glucagons promoter), neuronal specificity promoter (such as tyrosine hydrozylase promoter or neuronspecific enolase), endothelial cell specific promoter be (such as von Willebrand disease (von Willebrand); Please refer to Ozaki et al., Hum Gene Ther.7: 1483-1490,1996) and smooth muscle cell specificity promoter (such as 22a). The tumor cell specific promoter also can be used for treatment of cancer, and it comprises for the melanomatous EGFR-TK specificity promoter of B16 and (please refer to Diaz et al., J.Virol.72: 789-795,1998), the DF3/MUC1 that is used for some breast cancer (please refer to Wen et al., Cancer Res.53: 641-651,1993); For breast cancer, also can use the fatty specificity promoter of human aromatase cytochrome P450 (aromatase cytochrome P450, P450arom) (to please refer to US Patent No. 5446143; Mahendroo et al., J.Biol. Chem.268: 19463-19470,1993; And Simpson et al., Clin. Chem.39: 317-324,1993). The α-fetoprotein promoter can be used for indicating in the hepatoma to express (please refer to Chen et al., Cancer Res.55: 5283-5287,1995). When not needing or do not expect tissue specific expression, the n-3 desaturase coding sequence can place under the control of promoter (such as β-actin actin) of continuous activation (namely being connected to operably on the promoter of continuous activation). The promoter of other continuous activation is known by the personage of this area and is used.

Carrier of the present invention and other nucleic acid molecules (such as fat-1 cDNA itself) also comprise the temporary transient sequence of expressing of restriction transgenosis. For example, transgenosis can be by drug-induced property promoter control, for example this drug-induced property promoter comprises cAMP reaction component (CRE) enhancer and (please refer to Suzuki et al., Hum. Gene Ther. with the transfected or infected cell of cAMP regulating drug treatment in promoter7: 1883-1893,1996). Perhaps, inhibition sub-component (repressor element) can be suppressed at transcribing under the medicine existence and (please refer to Hu et al., Cancer Res.57: 3339-3343,1997). Also can be combined with the erg1 gene promoter by ionising radiation (radiotherapy) in order to do the space control of reaching expression. The body of constructing that comprises this adjusting sequence is in protection scope of the present invention.

Comprise the host cell (comprising expression vector) of described nucleic acid molecules also in protection scope of the present invention. This cell can be prokaryotes or eukaryotic cells. The prokaryote that is fit to comprises bacterial cell, and suitable eukaryotic cells comprises the cell of mammal, birds and fish. Cell line (comprising those foundation and be stored in the public database person) also can be used as host cell. Any cell in these cells all can be used on (but being not limited only to) and optimizes in the process of nucleotide sequence. This cell can be that healthy cell also can be the cell (for example, this cell may be suffered from inflammation or turn shape and/or propagation with the speed of not expecting (such as the height of not expecting)) with disease.

The albumen of described nucleic acid molecules and their codings thereof can be included in the medical composition. For example, said composition can comprise described expression vector and physiologically acceptable diluent (such as common salt solution or the physiologically acceptable buffer of phosphate buffered saline (PBS) for example). For the human individual, the method that yes adopts treatment or prevent, but the present invention is not limited only to this. Medical composition can be formulated into for throwing and gives the animal in domestic animal, pet, zoo or the circus troupe or the animal of the injured or ill found in the open air. No matter be which kind of individuality, this methods for the treatment of is thought successful, even can not eradicate potential disease or illness fully. The progress (such as neurodegenerative disease or cancer) of improving one or more symptom or the sign of disease or slowing down disease also all is useful, and can reach by using described composition. This nucleic acid molecules may be to exist with concentrated form or to be fit to throw the amount (as treating upper effective dose) of giving to individuality. Only have after offeing medicine to individuality, the n-3 PUFA that expression of nucleic acid n-3 dehydrogenase reaches cell raises and/or n-6 at intraindividual content: when the ratio of n-3 PUFA tended to balance to useful direction, it is upper effective that the amount of this dispensing just is considered to treatment.

The present invention also comprises the non-human transgenic animal (such as mammal, birds or fish) with described nucleic acid molecules. Such animal can be to entrust one's child to the care of sb., feed, catch or hunt the person that is used as food (as being consumed (such as domestic animal or pet) by human or other animal). Wherein, can be the mammal of ox, pig, sheep and so on; The birds of chicken, turkey, duck, goose or pheasant and so on; The fish animal of the class of salmon, trout, yaito tuna.

The food or the dietary supplement that comprise these non-human transgenic animals or tissue or its processing part are also included within protection scope of the present invention. This food may be to process without processing (such as the whole part (such as joint, dactylus or organ) of whole animal or animal) or the animal of certainly slaughtering or its part (such as bone, fat, skin or the animal oil that therefrom extracts). The method of making dietary supplement (such as fish-oil capsule) is to know in this area and can be applicable to gene modification animal of the present invention. The present invention also comprises the method for making food or dietary supplement in described animal (such as transgene mammal, birds or fish). Those methods can be reached by any way; As long as its source is or comprises such as the present invention and setting forth and the non-human transgenic animal (or its part) that produces.

Other method of the present invention comprises by give the content that above-mentioned food or dietary supplement improve n-3 fatty acid in the individual food to the individuality throwing.This individuality may look to be healthy or have been suffered from cancer (as breast cancer, colon cancer, prostate cancer, liver cancer, cervix cancer, lung cancer, the cancer of the brain, cutaneum carcinoma, cancer of the stomach, Head and Neck cancer, cancer of pancreas, leukemia or oophoroma) by diagnosis.Described composition also is used in individual interior by for example throwing the method that the described nucleic acid molecules that gives the treatment effective dose suppresses neuronal cell death to individuality.Therefore, this individuality may be diagnosed and suffer from neurodegenerative disease (as Alzheimer's, Parkinson's disease, Huntington).Other can be treated or preventible illness comprises deformity or tendency deformity, diabetes, obesity, cutaneous disorder, ephrosis, ulcerative colitis, Crohn's disease or the COPD of cardiac arrhythmia, angiocardiopathy, cancer, inflammation, autoimmune disease, retina or brain.This individuality can also be to have accepted or be about to the individuality that acceptance comprises the graft of biologic-organ, tissue or cell.The method that adopts is both can also can throw and described nucleic acid molecules to graft to individuality.

Be noted that described nucleic acid molecules (comprising the host that those use optimizing codon) can be used for producing non-human transgenic animal.Yet the present invention is not limited to this, we wish this nucleic acid be mainly used in raise or as the gene modification animal of food source.This animal can utilize the technology of knowing in the art to produce.More specifically, mammal and fish can obtain by using the method described in the following embodiment.

In specific embodiment, the present invention is to be feature with the transgenosis fish of the nucleotide sequence that includes the dehydrase of encoding or transgenic bird, and wherein this dehydrase can be converted into the dehydrogenation of n-6 fatty acid corresponding n-3 fatty acid.The transgenosis fish can be cod or any gadidae, Gadiformes fish; Halibut; Catfish or any Pacific herring order fish; Mackerel or any mackerel section fish; Salmon or any salmon fishes comprise trout; Perch or any bacalao; Westerly wind or any clupeidae fish; Ray or any Rajidae fish; Flatfish or any Osmeridae fish; Sole or any Soleidae fish; And yaito tuna or any mackerel section fish.Other fish such as above-mentioned and known by those skilled in the art scholar.

In producing transgenosis fish or birds process, can use any described nucleotide sequence, comprise the nucleotide sequence that has C.elegans fat-1 gene.Any nucleotide sequence can have at least one optimizing codon.For example, this sequence can comprise at least 5, maximum 150 optimizing codon.In specific embodiment, do not consider the primary source of desaturase coding sequence, the nucleic acid molecules that is used to produce transgenic bird or fish animal comprises 5-10,10-15,15-20,20-25,25-30,30-35,35-40,40-45,45-50,50-55,55-60,60-65,65-70,70-75,75-80,80-85,85-90,90-95,95-100,100-105,105-110,110-115,115-120,120-125,125-130,130-135,135-140,140-145,145-150,10-125,25-100,30-90,40-80,50-70 or general 60 optimizing codon.In one embodiment, the transgenosis that is positioned at fish or birds body can comprise the nucleic acid molecules that has nucleotide sequence shown in Figure 180.

Also show in addition, the position of this optimizing codon be used to produce constructing of non-human transgenic animal (mammal, birds and fish animal as described) also can be different in the body.For C.elegan fat-1 gene, optimizing codon can be in position 6,9,18,20,22,24,28-30,33-36,47,49,52,54,58,60,61,64,67,69-71,73,77,79,81,86,89,92,94-95,100,101,105,106,112,115,118,124,127,128,131,146,151,154,161,163,164,169,178,187,188,195,197,200,202,206,210,214,217,221,223,225,227,228,232,234,241,245,255,271,280-282,284,285,301,303,310,312,327, one or more position of 362 or 370.When the dehydrogenase coding genes that uses was not C.elegan fat-1 gene, codon optimized position also can be in these positions on one or more (comprising all) position.In gene of the same race (as the n-3 dehydrogenase gene of plant or fungi), the optimization position can be and top listed corresponding position.

In the embodiment that describes subsequently, evaluation to RNA analysis and enzyme can be assessed the gene expression effect, (these means all are routine techniques means in the present technique field, can be used to measure any variant of the bioactive fat-1 sequence of tool also can to use gas chromatography mass spectrometer to measure the fatty acid collection of illustrative plates; The mensuration that also can be used as the sample that in sick body body, extracts) with fat-1 expression.

What the study portion in following adopted is the cortex nerve.Fat-1 expresses and not only improves n-6: the collection of illustrative plates of eicosanoid in n-3 fatty acid proportion and the neuron, thus but also the opposing cell death improves the survival rate of cell.Detailed it, fat-1 expresses and improves fatty acid proportion and the Apoptosis that takes place lacking that protection rat cell opposing growth factor under the situation that external source n-3 PUFA replenishes is extracted out.Thus, described nucleic acid molecules (or other composition) can be used as neuroprotective agent, and can put into premature or the old patient (can also be animal body or the animal part tissue that is consumed by patient) who suffers from neurodegenerative disease.The gene transfer protection effect of opposing neuronal cell death is equal to the protection effect of replenishing n-3 fatty acid again.

Can cause retina and brain dysplasia owing to lack n-3 PUFA, especially (please refer to Uauy et al., Lipids the premature 36: 885-895,2001), and if lack n-3 PUFA in the animal body and will show the cognitive behavior and the visual loss that are losing one's memory, depend on unduly space and background and (please refer to Carrie et al., Neurosci.Lett. 266: 69-72,1999; Yehuda et al., J.Neurosci.Res. 56: 565-70,1999), so the positive result that the present invention obtains is especially exciting.And the disease situation on the generation various neurologys on mankind of showing is associated with n-3 shortage situation and (please refer to Vancassel et al., Prost.Leuk.Ess.Fatt.Acids 65: 1-7,2001; Hoffman and Birth, World Rev.Nutr.Diet 83: 52-69,1998).

Below the biological function of PUFA further is discussed, these functions are to influential according to the condition type of the therapeutic modality that adopts described nucleic acid molecules (and other composition).PUFA is the important composition composition of phospholipid cell membrane, and is that the precursor (precursor) that comprises the sender family of molecule (eicosanoid) of prostaglandin, thromboxane and leukotriene (please refer to Needleman et al., Ann.Rev.Biochem. 55: 69-102,1986; Smith andBorgeat, In Biochemistry of Lipids and Membranes, D.E.Vance ﹠amp; J.E.Vance, Eds., Beniamine/Cummings, MenloPark, CA, 00325-360,1986).The eicosanoid that derives from PUFA all plays key effect at adjusting inflammation, cytokinin release, immune response, platelet aggregation, vascular reactivity, thrombosis and allergic phenomena and (please refer to Dyerberg et al., Lancet 2: 117-119,1978; Cyerbergand Bang, Lancet 2: 433-435,1979; James et al., Am.J.Clin Nutr. 7: 343S-3438S, 2000; Calder, Ann.Nutr.Metab. 41: 203-234,1997).The main fatty acid precursor of this sender molecule is for providing the arachidonic acid (AA of the n-6 substrate of being responsible for the Synthetic 2 series compound, 20:4n-6), and be responsible for the arachic acid that the n-3 substrate of similar synthetic reaction takes place 3 series compounds and remaining double bond (EPA, 20:5n-3).The ratio of n-6 in the phosphatide: n-3 has been improved respectively from the balance between 2 series of AA and EPA and 3 serial eicosanoids.Eicosanoid from AA (2 series) and EPA (3 series) is obviously different on function, and some exists complete relative function (to please refer to Dyerberg et al., Lancet on physiology 2: 117-119,1978; Cyerberg and Bang, Lancet 2: 433-435,1979; James et al., Am.J.Clin Nutr. 7: 343S-3438S, 2000; Calder, Ann.Nutr.Metab. 41: 203-234,1997).3 serial eicosanoids are weak agonistic muscles, and under some situation, 2 serial eicosanoids are Opposing muscles.For example, 2 serial eicosanoids promote inflammation and platelet aggregation, and challenge, and 3 serial eicosanoids then improve these effects.In addition, PUFA (please refer to Price et al., Curr.Opin.Lipidol participate in the interchange of cell pair cell between gene expression and the cell with the free fatty acid form in the middle of 11: 3-7,2000; Sellmayer et al.Lipids 31Suppl:S37-S40,1996; VonSchacky, J.Lab.Clin.Med. 128: 5-6,1996).Like this, PUFA just can suppress various biological effect.

Described composition and method can be used for treating various particular cases, improve holistic health, and need not to consider its original condition (as unhealthy, general health or very healthy).The illness of any methods of treatment of giving n-3 PUFA by throwing all can be treated by the method that the gene (as C.elegans fat-1 gene) of coding n-3 dehydrase of the present invention is given in throwing.Part situation according to this methods of treatment can be described in detail in detail subsequently.

As medicine and nutritional supplementation food, n-3 PUFA has attracted suitable notice (to please refer to Connor, Am.J.Clin.Nutr. 70: 560S-569S, 1999; Simopoulos, Am.J.Clin.Nutr. 70: 562S-569S, 1999; Salem et al., Lipids 31: S1-S326,1996).In 25 years, surpassing 4500 research reports all is about the effect of exploitation n-3 fatty acid to human metabolism and healthy (as cardiovascular health) in the past.From epidemiology to cellular tissure and zooscopy have recognized that all that to randomized controlled trial the effect of omega-3 fatty acid (please refer to Leaf and Kang, World Rev.Nutr.Diet. 83: 24-37,1998; De Caterina et al., Eds., n-3 Fatty Acids and VascularDisease, Springer-Verlag.London, 1999, pp166; O ' Keefe and Harris, Mayo Clin.Proc. 75: 607-614,2000).Main beneficial effect has the minimizing that sudden death takes place (to please refer to Albert et al., JAMA 279: 23-28,1998; Siscovicket al., JAMA 274: 1363-1367,1995), reduce ARR danger (Kang and Leaf, Circulation 94: 1774-1780,1996), reduce that the triglyceride level (please refer to Harris, Am.J.Chin.Nutr. in the blood plasma 65: 1645S-1654S, 1997) and reduce blood clotting trend and (please refer to Agren et al., Prostagland.Leukot.Essent.Fatty Acids 57: 419-421,1997; Mori et al., Arterioscler.Throm.Basc.Biol. 17: 279-286,1997).Epidemiological study shows, another n-3 fatty acid, and α-linolenic acid can reduce miocardial infarction and (please refer to Guallaret al., Arterioscler.Thromb.Vasc.Biol. 19: 1111-1118,1999) and easily occur in women's fatal ischemic heart disease on one's body and (please refer to Hu et al., Am.J.Clin.Nutr. 69: 890-897,1999) danger.Nearest several randomized controlled trial shows that α-linolenic acid (please refer to de Lorgeril et al., Circulation 99: 779-785,1999) and ocean omega-3 fatty acid (please refer to Singhet al., Cardiovasc.Drugs Ther. 77: 485-491,1997; VonSchacky et al., Ann.Intern.Med. 130: 554-562,1999; GISSI-Prevenzione Investigators, Lancet 354: 447-455,1999) the coronary disease M ﹠ M of patients with coronary heart disease is all brought advantageous effects.N-3 fatty acid EPA in vivo or suffer from the animal experimental model of cancer and have anticancer function (to please refer to Bougnoux, Curr.Opin.Clin.Nutr.Metab.Care 2: 121-126,1999; Cave, BreastCancer Res.Treat. 46: 239-246,1997).Those absorptions that studies show that to the mankind are rich in that the cancered probability of crowd of EPA is obviously low (to please refer to Rose and Connolly, Pharmacol.Ther. 83: 217-244,1999).Additional n-3 PUFA shows has result of treatment (to please refer to Kremer, Am.J.Clin.Nutr. to for example arthritic inflammatory autoimmune diseases 71: 349S-351S, 2000; Ariza et al., Semin.Arthritis Rheum. 27: 366-370,1998; James et al., Am.J.Clin.Nutr. 71: 343S-348S), the non-human primate (be please refer to Neuringer et al., Proc.Natl.Acad.Sci.USA 83: 4021-4025,1986) and neonate (Uauy et al., Proc.Nutr.Soc. 59: 3-15,2000; Uauy et al., Lipids 31: S167-176,1996) the n-3 fatty acid DHA that studies show that be necessary for the normal function sexual development of retina and brain, especially to the premature.And, show that also n-3 PUFA also has very positive effect to solving some other clinical problem, (please refer to Appel et al., Arch.Intern.Med. comprising the image height blood pressure 153: 1429-1438,1993), diabetes (please refer to Raheja et al., Ann.N.Y.Acad.Sci. 683: 258-271,1993), obesity (please refer to Clarke, Br.J.Nutr. 83: S59-66,2000), cutaneous disorder (please refer to Ziboh, World Rev.Nutr.Diet. 66: 425-435,1991), ephrosis (please refer to De Caterina et al., Kidney Int. 44: 843-850,1993), ulcerative colitis (please refer to Stenson et al., Ann.Intern.Med. 116: 609-614,1992), Crohn's disease (please refer to Belluzzi et al., N.Engl.J.Med. 334: 1557-1560,1996), COPD (please refer to Shaharet al., N.Engl.J.Med. 331: 228-233,1994) and organ transplant rejection (please refer to Otto et al., Transplantation 50: 193-198,1990).Usually, the ratio of the n-3 of balance: n-6 is that normal development and growth are necessary in the body lipid cell, and plays an important role when suppressing and treating some clinical problems.Described disease, imbalance or situation need adopt the methods of treatment of described nucleic acid molecules (and other composition).For example, reach the purpose of treatment or prevent disease, imbalance and illness by offeing medicine to the individuality of the coding n-3 dehydrase expression vector of fat-1 gene order (as have).

Discover recently and (please refer to Simopoulos, Poultry Science 79: 961-970,2000), n-6: the ratio of n-3 fatty acid is greatly about 10: 1 to 20: 1.This studies show that the rely development and set up gene model (n-6/n-3=1: 1) (please refer to Leaf and Weber, AM.J.Clin Nutr. of the mankind of present western diet 45: 1048-1053,1987) required n-3 fatty acid will lack.Because n-6 and n-3 fatty acid all have tangible difference on metabolism and function, and very important complementation is arranged on physiologic function, so both balances are very important for homoiostasis and normal development.Yet, lack the n-3 dehydrase in the mammalian cell, it is not interconvertible causing n-3 and n-6 PUFA.Therefore, in biological cell n-6 and n-3 PUFA between balance rely on food supply to keep.By increasing the edible food of n-3 PUFA or the tissue concentration that the n-3 PUFA product that supplement the nutrients improve n-3 fatty acid in the mankind or the animal body of being rich in.In view of the potential therapeutic action of n-3 PUFA, the scientific worker advises should reducing in the diet one after another to the absorption of n-6 fatty acid and the absorption that increases n-3 fatty acid (please refer to Simopoulos, Food Australia in the world 51: 332-333,1999).The suggestion that nearest American Heart Association (American Heart Association) also makes on this diet (please refer to AHA Dietary Guidelines:Revision 2000, Circulation 102: 2284-2299,2000).

Though the edible n-3 PUFA product that supplement the nutrients are a kind of safe intervening acts, have many limitations.For example, n-3 PUFA tissue concentration has a substantial raising in the body in order to reach, and needs to continue at least individual month n-3PUFA high dose of 2-3, takes for a long time.Fatty acid biological drug effect rate from food to cell comprises digestion, absorption, transmission and metabolic a series of physiology course of fat.Like this, the edible efficient that gets involved can produce difference according to individual different physiology and health status.The patient who is in crisis situation or functions of intestines and stomach disorder can not picked-up or absorption fat food or n-3 fatty acid extra-nutrition product.In addition, capsule-type fish oil supplementation nutriment is because its calorie content is very high, and it is edible lifelong every day also to be not suitable for people.And picked-up may exist the toxic component that contains mercury or the danger of other organic toxin from the fish in the stretch of coastal water or lake, and effectively the food intervening act needs eating habit clocklike simultaneously, and this point is difficult to keep concerning some people.In sum, press for a kind of can be rapidly and improve effectively n-3 PUFA content in the cell and balance n-6: n-3 ratio and not by the method for long-term absorption fish or fish oil supplementation nutriment.Method of the present invention is by producing an alternative food source (by breeding transgenic livestock, its cell contains than the more n-3 PUFA of non-transgenic domestic animal) or satisfying this needs by throw the gene that gives coding n-3 dehydrase to sick body (as patient).The significant advantage of method of the present invention is that they not only improve the tissue concentration of n-3 PUFA, and has also reduced the excessive n-6 PUFA level of inner generation simultaneously.

Embodiment

Embodiment 1: make up recombined adhenovirus

The recombined adhenovirus that carries the fat-1 gene makes up according to the described step of the following Heet of being similar to al. (please refer to Proc.Natl.Acad.Sci.USA95:2509-2514,1998).N-3 fatty acid dehydrogenase cDNA (fat-1 gene) is so kind as to give by Dr.J.Browse (Washington State University) and (but also can uses in this area the synthetic or choosing of ordinary skill and information to grow and get among the pCE8; Please refer to Spychallaet al., Proc.Natl.Acad.Sci.USA 94:1142-1147,1997; U.S. Pat 6194167; And with reference to figure 17A and Figure 17 B).The cDNA of pCE8 inserts gene and shears out and be inserted into from plasmid the transfer vector (shutter vector) through the dual cleavage reaction of limiting enzyme EcoRI/Kpnl, carries out the recombined adhenovirus skeleton according to the method for He et al. (supra) then.Form two first generation 5 type recombined adhenovirus: carry the green fluorescent protein (GFP under the control of macrophage type virus (CMV) enhancer, as reporter gene) Ad.GFP, and carry fat-1 gene and GFP gene and the Ad.GFP.fat-1 under cmv enhancer control separately.As mentioned above, recombinant virus is bred in 293 cells and is become the virus (please refer to Hajjaret al.Circulatin 95:423-429,1997) of high titre value.This structure is sheared by enzyme and dna sequence analysis is determined.Equally with reference to Hajjaret al., Circulation 95:423-429,1997 and Hajjar et al., Circ.Res.81:145-153,1997.

Wild-type adenovirus infects can detected E1 sequence and the cytopathic effect of non-admissibility A549 cell line assessed by polymerase chain reaction (PCR).If desired or necessary, also availablely have other and strengthen self-control adenovirus vector or adeno-associated virus virus (adeno-associatedviral, AAV) carrier substitutes.

Embodiment 2: cardiac muscle cell's adenovirus is cultivated and is infected

Use cardiac muscle cell's extraction system (National CardiomyocyteIsolation System) (by Worthington Biochemical Corp., Freehold, NJ provides) cardiac muscle cell is extracted from the mouse that was born a day.Place 6 orifice plates then, in F-10 culture fluid (containing 5% hyclone, 30% horse serum, 37 ℃), place the tissue culture device (to contain 5%CO ₂With 98% to humidity) in cultivate.Cell as experiment need be through 2-3 days cultivation.In the culture fluid that contains 2% hyclone (FBS), add variable concentrations (5*10 ⁹-10 ¹⁰Pfu) virion.Cultivate after 24 hours, in culture fluid, replenish 18:2n-6 and the 20:4n-6 of 10 μ M.Infect after 48 hours, this cell can be used (as can be used for analyzing gene expression, fatty acid composition, survival rate or growing ability (as propagation or division rate)).

Embodiment 3: detect fat-1 expression and RNA analysis with fluorescence microscope

In the molecular biosciences field, there is the method for much knowing to detect gene expression.Here, among the cardiac muscle cell after above-mentioned infection step the expression of fat-1 adopt the vision-based detection of infection cell and the test of RNase enzyme protection to measure.

Know clearly it, adopt the GFP coexpression to discern infected cell and express transgenic cell.After infecting about 48 hours, nearly all cell (＞90%) demonstrates bright fluorescence, represents high gene transfer rate and high genetically modified expression (seeing also Fig. 1).The expression of Fat-1 genetic transcription also can be used RPA III ^TMEquipment (being provided by Ambion company) adopts RNase enzyme protection test method to measure.Letter speech, the step of drafting according to manufacturer use RNA extract equipment (providing) by Qiagen company with total RNA from through cultured cells, extracting.Like this, the plasmid pCE8 that has the fat-1 gene is linearized, and can be used as and transcribe template.Use in vivo ³³P-UTP transcribes antisense RNA probes, and total RNA that this antisense RNA probes warp extracts from the cardiac muscle cell is hybridized and sheared in order to do the RNA and the probe that will not have to hybridize are removed with the RNase enzyme.Shielded RNA:RNA carries out electrophoresis by denaturant gel to be separated, and carries out video picture according to autoradiography.The probe of the β-actin gene that points to is as control group.In the cell that infected by AD.GFP (yet as control group), do not find fat-1 mRNA, but in the cell that infected by Ad.GFP.fat-1, find a large amount of fat-1 mRNA (seeing also Fig. 2).This experimental result shows, gives very high fat-1 gene expression through the gene transfer that adenovirus is cultivated in the cardiac muscle cell who originally lacks the fat-1 gene.

Embodiment 4: lipid analysis; The effect that the n-3 dehydrase is formed fatty acid

Lipid analysis can be used to judge whether the fat-1 expression of gene is converted into n-3 fatty acid with n-6 fatty acid among cardiac muscle cell's (or other cell type), thereby changes fatty acid composition in the cell.During described cardiac muscle cell subsequently infected, cell was cultivated 2-3 days in the culture fluid that is supplemented with n-6 fatty acid (10 μ M 18:2n-6 and 10 μ M20:4n-6).Form according to the fatty acid of the total bioblast of foregoing analytical afterwards and (please refer to Kanget etal., Biochim.Biophys.Acta. 1128: 267-274,1992; Weylandt et al., Lipids 31: 977-982,1996).

With the chloroform/methanol that contains 0.005% butylated hydroxytoluene (as age resistor) (2: 1, volume ratio) solution lipid is extracted.Adopt 14%BF ₃/ methanol reagent prepares fatty acid methyl ester.The amount of utilization gas chromatography/mass spectrometry commercial measurement fatty acid methyl ester, used experimental instrument is HP5890 Series II gas chromatograph and the HP5971 mass spectrograph that Supelcowax SP-10 polarity capillary chromatographic column is housed.Syringe and detector respectively remain on 260 ℃ and 280 ℃.Baking oven program originally temperature is 150 ℃, kept 2 minutes, rises to 200 ℃, keeps 4 minutes with 10 ℃/min speed then, rises to 240 ℃, keeps 3 minutes with 5 ℃/min speed again, rises to 270 ℃, keeps 5 minutes with 10 ℃/min speed at last.Carry gas flow rate to remain 0.8mL/min.In molecular weight range 50-550AMUs, carry out total ion monitoring.By the relatively more different fatty acid zones of analyzing and internal standard fixed concentration zone, measure the fatty acid molecule mass number.

Be subjected to the fatty acid spectrogram of Ad.GFP cellular control unit that infects and the cell that infected by Ad.GFP.fat-1 that obviously different (seeing also Fig. 3) are arranged.And the cell that infected by Ad.GFP is compared in fatty acid spectrogram part as broad as long with the cell that does not infect.In the cell of expressing the fat-1 gene (n-3 dehydrase), the n-6 fatty acid major part of almost all kinds all is converted into corresponding n-3 fatty acid, and promptly 18:2n-6 is converted into 18:3n-3,20:2n-6 and is converted into 20:3n-3,20:3n-6 and is converted into that 20:4n-3,20:4n-6 are converted into 20:5n-3,22:4n-6 is converted into 22:5n-3.Thus, compare with the fatty acid of the cellular control unit that infected by Ad.GFP, the fatty acid composition of expressing the cell of fat-1 gene is effectively transformed (seeing also Fig. 5).Importantly, 1: 1.2 in the cell of expressing the n-3 fatty acid dehydrogenase is reduced in the ratio of n-6: n-3 15: 1 from cellular control unit.

Embodiment 5: measure the eicosanoid after fat-1 expresses

Because 20:4n-6 (AA) and 20:5n-3 (EPA) are respectively the precursors (precursor) of 2 series and 3 serial eicosanoids, the difference of AA and EPA inclusion may be brought the difference of eicosanoid product in the cell.Like this, only need to measure eicosanoid product in the infected cell, the preceding eicosanoid of measuring need start with calcium ion channel A23187 by the EIA instrument that uses the special detection PGE2, and wherein this PGE2 is with PGE3 16% cross reaction to be arranged.Know clearly it, use enzyme immune detection instrument (providing) to measure PGE2 by Assay Designs company according to the step that manufacturer is drafted.(16% cross reaction being arranged) with PGE3.Clean the cell of cultivating with the serum-free medium that contains calcium ion channel A23187 (5 μ M).Cultivated 10 minutes, this adjustment medium is restored, and then can be used for measuring eicosanoid.The amount of the PGE2 that is produced by cellular control unit is than the amount obviously high (seeing also Fig. 4) of the PGE2 that is produced by the cell of expressing through the n-3 dehydrase of fat-1 coding.

Embodiment 6: the analysis of zooblast in the culture

In present embodiment and next embodiment, we have set up three different experiments models: through cultured cells (will the obtaining test below through cultured cells of other type), adult rat and transgenosis mouse.Through the cultured cells model is to be used for characterizing the characteristic of enzyme and the biochemical effect of the mammalian cell n-3 dehydrase of expressing in vitro; Adult's rat model is to be used to assess the effect of the raising of the fat-1 gene through shifting n-3 PUFA tissue concentration in vivo; And the transgenosis mouse models is to be used to assess the influence of transgenosis to long-term, the system of various tissues or organ in the body.For two models in front, the method that shifts (intervention recombined adhenovirus) by adenovirus is incorporated into the fat-1 gene in mammalian cell/tissue.For last model, by the transgenosis microinjection is realized gene transfer in mouse fertilized egg.After the gene transfer, can adopt mRNA and/or analysis of protein method (for example reference example 3) to characterize the transgene expression spectrum, the biochemical effect of the fatty acid composition of cell or tissue is measured (for example reference example 4) by gaschromatographic mass spectrometry (GC-MS) technology used in conjunction, and eicosanoid is measured (for example reference example 5) by enzyme immune detection instrument.By relatively from the data of expressing the fat-1 cell with come self-infection that identical (or similar) arranged however the cellular control unit of virus untransfected fat-1 gene or the data of tissue are discerned the variation place.The end of the curve map that these research institutes get is that biochemistry changes in interior fatty acid composition of expression cell and the eicosanoid spectrogram.

The cultivation of in fact any cell (comprising human cell's strain) all can be infected recombined adhenovirus (Ad.GFP.fat-1 or Ad.GFP), and transgene expression subsequently can be assessed by RNA or analysis of protein.Experimentation and correlation technique are described in the above, and its main points are embodied among Fig. 6.Comprise that all kinds cell of cardiac muscle cell, nerve cell, liver cell, endothelial cell and macrophage all has been used for the research of n-3 fatty acid.

The cardiac muscle cell can cultivate (please refer to embodiment 2) according to above-mentioned method after purifying, can (please refer to In ADissection and Tissue Culture Manual of the NervousSystem according to Schousboe et al. from the biggest mouse of 1-5 just as cerebellar granule nerve cell and hepatocellular other cell type, Shabar et al., Eds., Alan R.Liss, New York, N.Y., pp.203-206,1989) described method cultivation.Comprise that breast cancer cell is that the human cell that is can or be supplemented with in the RPMI RPMI-1640 of 10% hyclone (FBS) at the MEN culture fluid, is positioned over 37 ℃/5%CO with the leukaemia ₂Cultivate in the incubator.

With virion with variable concentrations (as 2*10 ⁹-2*10 ¹⁰Pfu) join to contain in FBS or the 2%FBS culture fluid and realize cell infection.After cultivating in 24 hours, infect culture fluid and replace with common culture fluid (10%FBS).Infect after 48 hours, the gained cell can be used for gene expression analysis or fatty acid composition analysis.Transgenosis can adopt fluorescence microscope (putting into fluorescence labeling in transgenosis) (to see also embodiment 1 and Fig. 1; Similarly, this fluorescence labeling also can be can be by the detected antigen protein of fluorescence antibody) or the RNA test method of the standard of employing assess.Owing to can not exist in the cellular control unit under the fat-1 gene normal condition, therefore be not difficult to identify cellular control unit and expression have the fat-1 gene cell between the difference of fat-1 mRNA.

The n-3 dehydrase facilitates the two keys of n-3 to be incorporated in the n-6 fatty acid, in order to do make formation than precursor n-6 fatty acid have more many pair key n-3 fatty acid (as 18:2n-6 → 18:3n-3,20:4n-6 → 20:5n-3).The conversion ratio that is subjected to matter to be converted into product (at a certain amount of product of certain hour section formation) is considered to be directly proportional with the expression/activity of dehydrase.Therefore, the functional activity of enzyme can or be measured changing effect (output) by following method and weigh at the sample that obtains in animal body (as tissue samples) in cultured cells.

Employing is subjected to the fatty acid dehydrogenase test method of matter through the conduct of the n-6 of radioactive substance mark fatty acid: this test can be according to (please refer to Biochim.Biophys Acta. by Kang et al. 1128: 267-274,1992) the described step of drafting carries out.The various n-6 fatty acid that have a mark that will combine with bovine serum albumin(BSA) (BSA) in brief, (as [ ¹⁴C] 18:2n-6, [ ¹⁴C] 20:4n-6) join in the culture fluid of serum-free and and cultivated 4-6 hour with cell.Afterwards, cell and culture fluid are collected.Extract lipid and methylate methylate (asking for an interview following description).Be soaked with AgNO ₃Isolate the fatty acid methyl ester that has mark according to degree of unsaturation (as the double key number amount) on the silica gel tlc plate of solution.By comparing reference standard, can identify the fatty acid band that has different two keys.Scinticounting can determine to have the amount of the fatty acid of mark, and cellular control unit data that obtain and the data that the cell of expressing the fat-1 gene obtains are compared.

Adopt gc analysis fatty acid: analyze fatty acid with gas chromatograph-mass spectrometer and can measure fatty acid changing effect (asking for an interview following description) more accurately.If adopt this method, then do not need the fatty acid of radioactive substance mark.Select the different matter that is subjected to, content of fatty acid in the cultured cells of expressing the n-3 dehydrogenase gene is analyzed.Can determine the changing effect of every kind of fatty acid by the comparison cellular control unit and the fatty acid spectrogram of the cell of expressing the fat-1 gene.

The fatty acid composition of total bioblast or phosphatide can be analyzed according to above-mentioned method and (please refer to Kang et al., Biochim.Biophys.Acta. 1128: 267-274,1992; Weylandt et al., Lipids 31: 977-982,1996).Step is as described below:

Lipid is purified (also can consult embodiment 4): add 5ml chloroform/methanol (2: 1, volume ratio) solution and strict the stirring 1 minute of containing 0.005% butylated hydroxytoluene (as age resistor) in the cell precipitation thing through cleaning, place then under 4 ℃ and spend the night.Add 1ml0.88%NaCl solution and stirring again.The chloroformic solution that will contain lipid collects.Residue is purified in the 2ml chloroformic solution again.Compile this chloroformic solution and dry under nitrogen, be stored in the sealing test tube with-70 ℃ then.

(thin-layerchromatography TLC) separates lipid: the TLC plate was put into 100 ℃ of oven activated 60 minutes to adopt thin-layer chromatography.Before using solvent poured into and make its balance at least one hour in the TLC groove.Use solvent system to launch sample on the silica G plate and also continue 30-35 minute in order to do total phospholipids and triacylglycerol are therefrom separated, wherein this dicyandiamide solution is made up of ether/diethyl ether/acetate (volume ratio 80: 20: 1).Separating single phosphatide is on the silica gel H plate, and the solvent system of use is: chloroform/methanol/2-propyl alcohol/0.25%KCl/ triethylamine (volume ratio 30: 9: 25: 6: 18).With the colour developing of 0.01%8-phenylamino-1-naphthalene sulfonic aicd solution, the spectrogram that will contain lipid shows, and the gel piece of collecting each lipid part is in order to methylating.

Fatty acid methylization: use 14%BF ₃/ methanol reagent prepares fatty acid methyl ester.In the teat glass that has the lid of using teflon seal, in the lipid sample, add 1ml or 2ml hexane solution and 1mlBF ₃/ methanol reagent.Sample after with nitrogen wash in 100 ℃ of heating one hour down, be cooled to room temperature and after adding 1ml water methyl esters from the hexane liquid phase, separate.Allow sample leave standstill 20-30 minute, the upper strata hexane layer is removed the back under nitrogen, concentrate in order to gas-chromatography (GC) analysis.

Gas chromatography-mass spectrometry analysis: reorganization is through methylated sample in 100-200 μ l hexane or isooctane solution, and 1-2 μ l wherein will be used for gas chromatographic analysis.In Hewlett-Packard 5890A gas chromatograph (by Hewlett-Packard, Avondage, PA provides), adopt (30 meters of Omegawas chromatographic columns; By Supelco, Bellefonte, PA provides).The gas of carrying secretly is hydrogen (2.39ml/min), and the split ratio of injection is 1: 31.Initial temperature is 165 ℃, continues 5 minutes, is elevated to 195 ℃ with 2.5 ℃ of/minute speed then, is elevated to 165 ℃ with 27.5 ℃/minute again.By with the fatty acid standard (by Nu-Chek-Prep, Elysian, MN provides) relatively can identify absworption peak, use Perkin-Elmer M1 planimeter (by Perkin-Elmer then, Norwood, CT provides) analyze all absworption peak area occupied ratios.These analytical methods all are different to all saturated fatty acids from C14 to C25 carbochain, mono fatty acid, bis-fatty acid and polyunsaturated fatty acids.Sample size calculates with outside specification.And, for the operation ionization voltage that carries out the Hewpett-Packard proton selection detector (model is 5972) that gas chromatography-mass spectrometry analysis adopted is 70eV, sweep limits 20-500Da.New absworption peak and the standard absorption peak in the NBS75KL database (please refer to National Bureau of Standards) that mass spectrogram is obtained (please refer to NuChek Prep, Elysian, MN) comparison.

Embodiment 7: n-3 dehydrogenase gene transfer effect in the assessment body

Experiment described here is incorporated into the fat-1 gene in animal tissue or the organ (as heart), and wherein the enzyme product is optimized the fatty acid composition rapidly by the amount that the amount that increases n-3 PUFA reduces n-6 PUFA.Select for use heart as the gene transfer subjects, because it was studied in numerous relevant experiments of n-3 fatty acid, and it also is vital organ.

Feed bimestrial ripe rat with common food or the food that is rich in n-6 PUFA and injected the adenovirus that carries the adenovirus of fat-1 gene (Ad.GFP.fat-1) or carry indicator GFP (Ad.GFP organizes gene in contrast) arbitrarily.These adenovirus will be sent in the heart of live body by microcatheter technology, the expression pattern of generation in whole heart all be homogeneity (please refer to Hajjar et al., Pro.Natl.Acad.Sci.USA 95: 5251-5256,1998).Infect (gene transfer) after two days, four days, ten days, 30 days and 60 days, animal dead collects their heart being used to and evaluates gene expression and analyze fatty acid and form.Another group mouse was fed two months with being rich in n-3 fatty acid (low n-6/n-3 ratio), and did not carry out gene transfer, with for referencial use.These experiments (wherein collecting sample with the different things nursing and in different time points) are to be used to judge whether the fat-1 gene transfer can cause that the biochemical effect of expectation (is the n-6/n-3 ratio, the eicosanoid collection of illustrative plates), and whether this biochemistry benefit is similar to or even be better than the effect that food intake (as n-3 PUFA fill-in) brings, how soon the substantial variation that obtains the fatty acid composition has, and how long this variation can continue.The rat of injecting signal (GFP) gene is considered control group (preliminary study shows that the gene transfer of GFP is to not influence of fatty acid composition).The experiment flow chart as shown in Figure 7.

Animal and food: the ripe Sprague-Dawley rat that quality is suitable is divided into three groups arbitrarily.Feed one of following three kinds of different foods for every group: common (substantially) food, be rich in n-6 food or be rich in n-3 food.According to these foods of preparation as described below.

Basic food: the fat-free food of a kind of muroid on sale on the market (by Agway Inc.C.G., Syracuse, NY provides), wherein added 2% (mass percent) corn oil; Be rich in n-6 food: in basic food, replenish 13% (mass percent) corn oil or safflower oil (being rich in n-6 fatty acid) again, contain 15% fat in the final food; Be rich in n-3 food: in basic food, replenish 13% (mass percent) fish oil (65% all is n-3 PUFA for 30%EPA, 20%DHA) (by PronovaBiocare A/S, Oslo provides) again, contain 15% fat in the final food.This group will be organized in contrast for this research.

Each week of food prepares an aliquot, is stored under-20 ℃, melts required amount every day.For preventing the oxidation of polyunsaturated fatty acids long-chain, add Vitamin E (100mg/100g fat) and butylated hydroxytoluene (BHT) (ultimate density is 0.05%) (BHT can prevent the autoxidation of unsaturated fatty acid in food preparation and deposit process).For guaranteeing that animal receives enough nutrition, the mouse in each group all need be weighed weekly.After feeding in eight weeks, animal begins receptor gene and shifts.

Adenovirus is transmitted record: adopt to be similar to by the described microcatheter technology of Hajjar et al. (supra) and realize adenovirus is delivered in the heart.In brief, with intranodal peritonaeum amobarbital (60mg/kg) anesthesia mouse, and place on the ventilation blower.Pass the not busy position of the 3rd rib from the left side, thoracic cavity and enter the thoracic cavity.Open pericardium, place the 7-0 suture at the left ventricle top end.Can see sustainer and pulmonary artery.To contain 200 μ l adenovirus (9-10*10 ¹⁰Pfu/ml) (left ventricle, LV) top is inserted into aortic root to 22 chi long ducts from left ventricle.Be clamped in sustainer and pulmonary artery, then solution injected from a conduit side far away.Continue to clamp for 10 seconds, make heart in closed system, aspirate (wait and hold ground) simultaneously.After 10 seconds, unclamp the anchor clamps that are clipped on sustainer and the pulmonary artery, close the thoracic cavity, conduit is removed, put back to cage.

After the gene transfer two days, four days, ten days, 30 days and 60 days, animal dead was removed and is infected virulent heart, with perfusion of saline or clean bloodstain, rapidly its portion of tissue was freezingly down measured in order to lipid analysis and eicosanoid at-80 ℃.Remaining tissue will be used to judge the protein level of mRNA level and/or n-3 dehydrase.

The high concentration adenovirus that other organ (for example brain, liver) also may be entered into blood infects.Therefore, other organ that comprises heart also will collect as transgene expression and lipid atlas analysis.

The method that cell part after cultivating recited above is adopted also will adopt in the present embodiment, comprise that evaluation (by RNA trace, the test of RNase albumen or hybridization in situ experiment), fatty acid composition analysis, the eicosanoid of transgene expression measured and statistical analysis technique.

Embodiment 8: transgenic animal

Described research is to produce transgenic animal of expressing the fat-1 gene and tissue and the organ lipid collection of illustrative plates that characterizes these animals.The transgenosis mouse has become estimates the model that get a good eye value of vivo gene in physiological importance.Can work out the transgenosis influence of various cell types in animal dis current different phase by research transgenosis mouse.This n-3 transgenosis mouse models will grow and cell biology in reach and grow from this commentaries on classics that derived compounds provides new chance the mouse for making the n-3PUFA that therefrom produces clear.

Express the transgenic animal of fat-1 gene for producing the energy overall situation, can use the chicken class β-actin promotor (CAG promotor) that contains the fat-1 expression carrier and contain cmv enhancer, these materials activity in most of cell type is all quite big, (please refer to Niwa et al. with regard to helping obtaining extensive and high-caliber transgene expression like this, Gene 108:193-199,1991; Okabe et al., FEBS Lett. 407: 313-319,1997) (following) to the special description of genetically engineered fish class do.Expression is constructed body micro-injection in unicellular embryo's protokaryon of C57BL/6X C3H mouse, to produce the transgenosis mouse.They will be fed, and form trangenic mice system.Weanling mouse is with basic food or be rich in the nursing of n-6 food.Collect each tissue from the mouse of all ages and classes (neonate, children's-1 month, grew up-6 months and old-12 months, each time period is chosen 3-5 mouse) to be used to assess transgene expression level and judgement fatty acid composition.Also to measure the level of eicosanoid in each tissue of blood plasma neutralization.Will be with one group of wild type mouse (C57BL/6) that same food (common food or be rich in n-6 food) is fed as control group.Experimental result will compare with the experimental result of the wild type mouse of feeding same food.This comparison step illustrates in Fig. 8.

Employing is similar to the method for being described by Okabeet al. (supra) and prepares metastatic gene.In brief, amplify the cDNA of coding fat-1 gene with primer PCR (5-agaattcggcacgagccaagtttgaggt-3 ' (SEQ ID NO:1) and 5 '-gcctgaggctttatgcattcaacgcact-3 ' (SEQ ID NO:2)), use pCE8-fat1 (providing) as model by Dr.J.Browse from Washington State University.Dna sequence dna has determined the PCR product.Be included in the EcoR1 of PCR primer and Bgl-II and be used to the fat-1 cDNA through amplifying is incorporated in the pCAGGS expression vector and go, chicken class β-actin promotor and macrophage virus enhancer, β-actin intron and bovine globin adenylate signal (providing by the Dr.J.Miyazaki from OsakaUniversity Medical School) wherein are provided in this pCAGGS expression vector.The promotor of inserting and coded sequence will introduce BamHI and Sa/1 restriction enzyme site, and through gel-purified.

Inject purified BamHI and Sa/1 fragment generation trangenic mice system to C57BL/6X C3H fertilized egg.The ovum transfer that is injected with DNA is interior to produce the transgenosis mouse to false pregnancy mouse (B6C3F1) body.Utilization PCR and tail southern blotting technique hybridization (Southern blot) analysis are discerned the head that feeds with the C57BL/6J mouse and are built the transgenosis mouse.Select to use their offspring's (no matter being heterozygote or homozygote) according to the expression of transgenosis or phenotype.

With common food or be rich in N-6 PUFA food and feed weanling transgenosis mouse (as mentioned above).Animal will be collected various tissues in order to measuring genetically modified expression and judging the fatty acid composition in all ages and classes (neonate, children's-1 month, grew up-6 months and old-12 months, each time period is chosen 3-5 mouse) death.Experimental result compares with the experimental result of the wild type animal of feeding same food.

The method that cell part after cultivating recited above is adopted also will adopt in the present embodiment, comprise that evaluation (by RNA trace, the test of RNase albumen or hybridization in situ experiment), fatty acid composition analysis, the eicosanoid of transgene expression measured and statistical analysis technique.

With foregoing theoretical basically identical, cultivated the transgenosis mouse of expressing C.elegans fat-1 gene, wherein C.elegans fat-1 gene code n-3 fatty acid dehydrogenase.These mouse can produce n-3 fatty acid from n-6 fatty acid, and the n-3 content of fatty acid increases in nearly all organ and tissue (for example lean meat or milk) in order to do making, the n-6 content of fatty acid reduces, and need not take in the food that contains n-3 fatty acid.This achievement has been supported our viewpoint, be that these class transgenic animal (as: but any animal of effective expression or overexpression fat-1 nucleotide sequence) are unique research tools, and be the novel and desirable n-3 fatty acid source that can be used for satisfying human or other Animal nutrition needs.

For reaching the purpose of heterogenous expression C.elegans n-3 fatty acid dehydrogenase in mouse body, by optimizing codon in the mammalian cell, being connected with macrophage virus enhancer (activity is all quite big in most of cell type) with chicken class β-actin promotor, (please refer to Niwa et al., Gene from the encode fat-1 gene of this albumen of original form modification 108: 193-199,1991; Okabe et al., FEBS Lett. 407: 313-319,1997).

Build the expression of fat-1 among mouse and offspring's thereof the F1 F1 from transgenosis head with the analytical method check of the Real-Time PCR of rear DNA and rear lipid.It is also very healthy that these transgenosis mouse look very normal.Lasting usefulness is rich in omega-6 fatty acid (major part is a linolenic acid) and contains a small amount of omega-3 fatty acid (account for greatly additional total fat content 0.1%) and feed transgenosis and wild type mouse.The food of feeding this n-3 shortage can make us be easy to discern their phenotype.Under this raising method, the wild type mouse does not have little or no n-3 fatty acid owing to self can not make from n-6 fatty acid generation n-3 fatty acid in they tissues, if and fat-1 transgenosis mouse in vivo producer shift, the n-3 fatty acid (from n-6 fatty acid) of a great deal of then should be arranged in their tissue.

Because the phenotype of trangenic mice system mainly by the reflection of lipid collection of illustrative plates, therefore only needs to use gas chromatograph-mass spectrometer that the fatty acid composition of the various organs of all ages and classes transgenosis mouse is analyzed.

What is interesting is that the muscle of transgenic animal changes maximum, shows that enzymic activity is the highest in this tissue in these ratios.Up to now, four generation trangenic mice systems (being homozygote or heterozygote) are all through check, and their fatty acid spectrogram all shows and continue high-caliber n-3 content of fatty acid, and functional activity is big and can transmit in vivo to show this transgenosis.The clear trangenic mice of expressing the fat-1 gene that shows of data can produce n-3 fatty acid from n-6 fatty acid, is rich in n-3 fatty acid in order to do making in their organ-/ tissue, and need not takes in the n-3 fill-in, and this is impossible take place in the wild type mammal.

These are discovered to producing the food (as lean meat, milk or egg) that is rich in n-3 PUFA provides a kind of new method, promptly cultivates big transgenic animal (as ox, pig, sheep, goat, rabbit, deer, chicken or other poultry (as duck, goose, pheasant and pheasant)) and/or transgenosis fish or other farm and cultures or be grown in river, lake, brook or the big marine edible animal that has the n-3 dehydrogenase gene.

The method that is used to cultivate these animals is done further more detailed narration below.

Expression vector: (please refer to Spychalla, et al., Proc.Natl.Acad.Sci.USA from C.elegans 94: 1142-1147,1997) on the basis of the fat-1 sequence of duplicating, use the mouse codon frequency simultaneously as a reference by the modification codon, the cDNA of composite coding n-3 fatty acid dehydrogenase.Fat-1 cDNA after synthetic confirms through the DNA ordering, copies to then in the pCAGGS expression vector that contains chicken class β-actin promotor and macrophage virus enhancer, β-actin intron and bovine globin adenylate signal.Insert promotor and coded sequence and will introduce Ssp I and Sfi I restriction enzyme site, and through gel-purified.

Cultivate trangenic mice: inject purified Ssp I and Sfi I fragment generation trangenic mice system to C57BL/6X C3H fertilized egg.The ovum transfer that is injected with DNA is interior to produce the transgenosis mouse to false pregnancy mouse (B6C3F1) body.The Real-Time PCR of utilization rear DNA and the analytical method of rear lipid are discerned the head that feeds with the C57BL/6J mouse and are built the transgenosis mouse.We obtain seven head and build the transgenosis mouse.Wherein three are used as breeding transgene mouse system.So far, each has bred the three generations.

Animal feeding: transgenosis mouse and wild mouse (C57BL/6J) are adhered to the safflower oil that contains 5% (percentage by weight), based on the rodent food of AIN-76A.In the safflower oil fatty acid composed as follows shown in: 10% saturated fatty acid, 14% unsaturated fat acid monomers, 76%n-6 polyunsaturated fatty acids and the unsaturated α-linolenic acid of about 0.1%n-3 poly.

Lipid analysis: (please refer to Kang et al., Biochim.Biophys.Acta. according to foregoing method 1128: 267-274,1992) analyzing total organizes the fatty acid of plasmid to form.With the chloroform/methanol that contains 0.005% butylated hydroxytoluene (as age resistor) (2: 1, volume ratio) solution lipid is extracted.Adopt 14%BF ₃/ methanol reagent prepares fatty acid methyl ester.The utilization gas chromatographic technique is analyzed fatty acid methyl ester, and used instrument is for being equipped with the full-automatic HP5890 system of flame ionization detector (flame-ionizationdetector).Chromatograph adopts Omegawax 250 capillary chromatographic columns (30m*0.25mmI.D.).Baking oven program originally temperature is 180 ℃, kept 5 minutes, rises to 200 ℃, keeps 48 minutes with 2 ℃/min speed then.By with the fatty acid standard (by Nu-Chek-Prep, Elysian, MN provides) relatively can identify absworption peak, use Perkin-Elmer M1 planimeter (by Perkin-Elmer then, Norwood, CT provides) analyze all absworption peak area occupied ratios.Compare by area and can determine the fatty acid quality the external perimysium reference of the fatty acid area of various analyses and fixed concentration.

Table 1. is wild type mouse (WT) and fat-1 transgenosis mouse (TG) ^*Between various organs and the n-6/n-3 ratio of tissue and the comparison of AA/ (EPA+DPA+DHA) ratio

	Omega-6/Omega-3 *		AA/(EPA+DPA+DHA)
						WT	TG	WT	TG
Muscle	49.0	0.7	11.3	0.4
					Milk **	32.7	5.7	15.7	2.5
Erythrocyte	46.6	2.9	27.0	1.6
					Heart	22.8	1.8	14.3	0.9
Brain	3.9	0.8	3.6	0.7
					Liver	26.0	2.5	12.5	0.9
Kidney	16.5	1.7	11.9	1.2
					Lung	32.3	2.2	19.8	1.2
Pancreas	23.8	2.4	17.3	1.5

^*Wild type and transgenosis mouse are all 8 all big female mouse and the identical foods of feeding

^*The n-6/n-3 fatty acid proportion is (18:2n-6+20:4n-6+22:4n-6+22:5n-6+)/(18:3n-3+20:5n-3+22:5n-3+22:6n-3)

Embodiment 9: suppress nerve cell death

Make up recombined adhenovirus (Ad): make up the recombined adhenovirus (Ad) that carries the fat-1 gene according to aforesaid method and (please refer to Kang et al., Proc.Natl.Acad.Sci.USA 98: 4050-4054,2001; Also can consult noted earlier).Used n-3 fatty acid dehydrogenase cDNA (fat-1 gene) from lipid pCE8 as previously mentioned.The cDNA of pCE8 inserts gene and shears out and be inserted into the pAdTrack-CMV carrier from plasmid through the dual cleavage reaction of limiting enzyme EcoRI/KpnI.Recombined adhenovirus skeleton carrier (pAdEasy) is grown product to produce two kinds of choosings in homologue then: carry the green fluorescent protein (GFP under the control of macrophage type virus (CMV) enhancer, as reporter gene) Ad.GFP, and carry fat-1 gene and GFP gene and the Ad.GFP.fat-1 under cmv enhancer control separately.Recombinant adenoviral vector DNA shears out from PacI.Linearized vector DNA sneaks into SuperFect ^TMBe used to infect 293 cells after (providing) by QIAGEN.This structure shears (as spacing figure) by enzyme and dna sequence analysis is determined.Check purified virus and confirm the sequence that this is viral again with the pcr analysis method.

Tissue culture and adenovirus infection: prepare mouse cortex nerve cell with standard technique.In brief, dissolving embryo's in utero 17 days (E17) mouse cortex nerve cell is then with 2*10 ⁶The cells/ hole is tiled in the hole that is coated with polylysine.At Neurobasal ^TMMedium (NBM, Life Technologies) cultured cell in the culture fluid, wherein be supplemented with in this culture fluid 25mM glutamic acid (Sigma Chemical Co., St.Louis, MO), 0.5mM glutamine, 1% antibody-antienzyme bacterium solution and 2% B27 (Life Technologies).Culture remains under 37 ℃, CO in the air ₂Concentration 5%, relative moisture 98%.Culture fluid changed once every 4 days.Cultivate after 8-10 days, cell transfecting has Ad-GFP (control group) or Ad-GFP-fat-1 lipid.In culture fluid, add bacteria particles to realize bacterial infection.Cultivate after 48 hours, cell is used for analyzing gene expression, fatty acid composition, eicosanoid product and inducing cell death.

RNA analyzes: the Fat-1 gene expression dose can be by using RPA III ^TM(provided by Ambion company, Austin TX) adopts RNase enzyme protection test method to detect mRNA and transcribes and measure equipment.In brief, the step of drafting according to manufacturer use total RNA separation agent (TRIzol, GIBcoBRL) with total RNA from through cultured cells, extracting.Like this, the plasmid pCE8 that has the fat-1 gene is linearized, and can be used as and transcribe template.Use ³³P-UTP, T7 polymerase (Riboprobe System ^TMThe T7 instrument is provided by Promega) transcribe antisense RNA probes in the body, this antisense RNA probes through extracting from the cardiac muscle cell total RNA (15 μ g) hybridization and shear in order to do the RNA and the probe that will not have to hybridize are removed with the RNase enzyme.Shielded RNA-RNA is dissolved in the sex change 5% sequence gel, and carries out video picture according to autoradiography.The probe of the β-actin gene that points to is as control group.In the cell that infected by AD.GFP (yet as the control group gene), do not find fat-1 mRNA, and in the cell that infected by Ad.GFP.fat-1, find a large amount of fat-1 mRNA.

Also can check described cell by fluorescence microscopy.Owing to express the infected cell and the GFP coexpression of fat-1 gene, so just be easy to identify these cells.Infect after 48 hours, the nerve cell of 30-40% is infected and is expressed by GFP.These results show, are originally lacking in the mouse cortex nerve cell of fat-1 gene, and the gene transfer that adenovirus Ad gets involved the back generation brings the height of fat-1 gene to express.

Lipid analysis: according to Kang et al. (Proc.Natl.Acad.Sci.USA 98: 4050-4054,2001) fatty acid is formed in the analyzing total bioblast.With the chloroform/methanol that contains 0.005% butylated hydroxytoluene (as age resistor) (2: 1, volume ratio) solution lipid is extracted.Adopt 14%BF ₃/ methanol reagent prepares fatty acid methyl ester.The amount of utilization gas chromatography/mass spectrometry commercial measurement fatty acid methyl ester, used experimental instrument is HP5890 Series II gas chromatograph and Supelcowax SP-10 polarity capillary chromatographic column (Supelco, Bellefonte, HP5971 mass spectrograph PA) is housed.Syringe and detector respectively remain on 260 ℃ and 280 ℃.Baking oven program originally temperature is 150 ℃, kept 2 minutes, rises to 200 ℃, keeps 4 minutes with 10 ℃/min speed then, rises to 240 ℃, keeps 3 minutes with 5 ℃/min speed again, rises to 270 ℃, keeps 5 minutes with 10 ℃/min speed at last.Carry gas flow rate to remain 0.8mL/min.In molecular weight range 50-550AMUs, carry out total ion monitoring.By the relatively more different fatty acid zones of analyzing and internal standard fixed concentration zone, measure the fatty acid molecule mass number.

The expression of Fat-1 makes n-6 fatty acid be converted into n-3 fatty acid, thus n-6: the ratio generation great change of n-3 fatty acid.The fatty acid spectrogram that cellular control unit obtains and infect spectrogram that the Ad-GFP-fat-1 cell is arranged between a great difference (seeing also Fig. 9 and Figure 10) is arranged.When infection had the cell of Ad-GFP to compare with the cell that does not infect, fatty acid composition did not change.And in the cell of expressing the n-3 dehydrase, the n-6 fatty acid of almost all kinds all is converted into corresponding n-3 fatty acid, and promptly 18:2n-6 is converted into 18:3n-3,20:4n-6 and is converted into that 20:5n-3,22:4n-6 are converted into 22:5n-3,22:5n-6 is converted into 22:6n-3.This in the cell of expressing the fat-1 gene variation of aliphatic acid composition make 1.7: 1 of the ratio of n-6: n-3 reducing in the cell of expressing n-3 dehydrase at 6.4: 1 in cellular control unit.The expression of C.elegans n-3 fatty acid dehydrogenase makes that also the DHA contents level is significantly increased in the transfectional cell.The raising of EPA and ALA level is accompanied by the minimizing of AA and LA, product P GE ₂Minimizing be by n-6: the change of n-3 fatty acid proportion and the intervention of DHA cause the inhibitory action of AA hydrolysis.

Measure eicosanoid: nerve cell death is relevant with 2 serial eicosanoids in age related neural degenerative disease and for example ischemic acute excitement murder by poisoning damage (please refer to Sanzgiri et al., J.Neurobiol.41:221-229,1999; Drachman and Rothstein, Ann.Neurol.48:792-795,2000; Bezzi et al., Nature391:281-285,1998).Arachidonic acid (AA, 20:4n-6) and arachic acid (EPA 20:5n-3) is 2 series and the precursors (precursor) of 3 serial eicosanoids respectively.Whether can bring the difference of eicosanoid product in the cell for the difference of judging AA and EPA inclusion, then need measure eicosanoid product in the infected cell, the preceding eicosanoid of measurement need start with calcium ion channel A23187 by the EIA instrument that uses the special detection PGE2.Know clearly it, use enzyme immune detection instrument (providing) to measure PGE2 by Assay Designs company according to the step that manufacturer is drafted.(16% cross reaction being arranged) with PGE3.Clean the cell of cultivating with LH buffer (containing 1%BSA), then this cell is put into the same buffer that contains calcium ion channel A23187 (5 μ M) and cultivated.Cultivate after 10 minutes, recover this adjustment medium, be used to measure eicosanoid then.Hang down 20% (see also Figure 11) through the amount of the PGE2 of the cell generation of the n-3 dehydrase of fat-1 coding than the amount of the PGE2 that produces by cellular control unit by expression.

Inducing cell death and growth of judgement cell and survival ability: by extracting the growth factor-induced cell death.The neuron transfection is after 48 hours, with culture fluid change into be supplemented with 25mM glutamic acid (provide by Sigma Chemical Co., St.Louis, MO) and the Neurobasal of 0.5mM glutamine ^TMThe Medium culture fluid.After growth factor is extracted 24 hours, use Vybrant ^TM(provided by Molecular Probes, Eugene OR) measures cytotoxic to Apoptosis Assay.The letter speech, clean cell with ice-cold phosphoric acid buffer saline solution (PBS), then this cell is sneaked among the ice-cold PBS that contains Hoechst 33342 solution (1ml/ml) and PI solution (1ml/ml) and cultivated 20-30 minute.When closing to an end, claps cultivation stage a photo.

Cell growth and survival ability: judge cell growth and survival ability with MTT cell proliferation equipment (providing) by Roche Diagnostic Corporation.Every well adds the reagent (100 μ l) that posts the MTT label.Cultivate after 4 hours, every well adds in the 1.0ml solvent soln.Then, cell is cultivated overnight down at 37 ℃, measures the solution absworption peak at 600nm place with spectrophotometer.

The Fat-1 expression of gene can protect mouse cortex nerve cell to avoid cell death forcefully.Behind the inducing cell death 24 hours, the cortex dyeing liquor that contains Hoechst33625 and PI shows that infection has the dyeing liquor of Ad-GFP-fat-1 to be subjected to cell death has the dyeing liquor of Ad-GFP to lack than infecting.MTT the analysis showed that the Ad-GFP-fat-1 cell survival can the force rate infection have the cell of Ad-GFP to improve greatly (p＜0.05) (seeing also Figure 12).These results show that all the expression of fat-1 can suppress nerve cell death and promote the cell survival ability.C.elegans n-3 fatty acid dehydrogenase can suppress nerve cell death and show n-6 in neuro-protective more: the importance of n-3 fatty acid proportion.Thus, provide n-6 in a kind of rapid statocyte with having the technology that active variant passes to nerve cell on fat-1 sequence or its physiology: the n-3 fatty acid proportion, change eicosanoid collection of illustrative plates (and the effect that nerve cell is produced the opposing cell death) and the method that need not replenish exogenesis n-3 PUFA.Interfere with food and to compare, this method is at balance n-6: more effective on the n-3 ratio, this be because it in the tissue concentration that improves n-3 PUFA, also reduced in the contents level of former n-6 PUFA.This method is to optimize the novel and effective method of fatty acid composition in the nerve cell, and it can be used as unique gene therapy or complementary therapy or chemoprophylaxis operation (for example being used for stroke patient).

Data analysis, statistical analysis: use Student-test pair cell survival ability data (MTT), fatty acid is divided into and the eicosanoid level compares.This analysis comprises 6 well/groups (removing 4 well/groups of lipid analysis), and every group of experiment repeats 3 times.The horizontal p of validity＜0.05.

Embodiment 10: the expression of fat-1 and inflammation-inhibiting in the human endothelium

For judging that n-6 is converted into n-3 PUFA and whether can applies to the human blood vessel endothelium of most of primary and whether can be used for studying the latent effect that prevents the endothelium activation after cell factor stimulates from the genetics angle, need to make up the first generation (type 5) recombinant adenoviral vector (Ad), this carrier contains a series of fat-1 transgenosiss that have the GFP expression casette that are under cmv enhancer (Ad.fat-1) control.GFP/ β-gal adenovirus is organized carrier (Ad.GFP/ β-gal) in contrast.Monolayer primary Human umbilical vein endothelial cells (HUVECs) was infected 36 hours with Ad.fat-1 or control group A d, be exposed in the 10mM arachidonic acid solution 24 hours then, subsequently by gas chromatograph accept lipid analysis, by enzyme immune detection instrument accept the surface adhesion analysis of molecules and by the technical research of video micrometering under the laminar flow conditions and monocytic series THP-1 between the interaction of endothelium.

The expression of fat-1 has changed lipid composition in the human endothelial cell greatly, and also with n-6: the ratio of n-3 PUFA becomes 1.4 from 8.5.And, being exposed to cell factor (TNF-α, 5 μ/ml, 4 hours) afterwards, the expression of fat-1 has significantly reduced expression surface adhesion molecule and inflammation sign (42%E-Selectin, 43%ICAM-1, and 57%VCAM-1 (p＜0.001)).

Whether the variation of having studied the adhesion molecule collection of illustrative plates then is enough to change at the most ubiquitous leucocyte endothelial mononuclear cell in atherosclerotic lesion place.Under the shear stress of laminar flow and about 2 dynes/cm2, the cohesive force that the HUVEC that infected by fat-1 brings is littler by about 50% than the HUVEC that infected by the control group carrier, and the THP-1 cell between almost not have the active force of mutual rotation.Like this, the heterogenous expression fat-1 gene of C.elegans dehydrase is given the ability that human endothelial cell is converted into n-6 PUFA n-3 PUFA.This ability has stoped the cytokine induction of endothelium inflammatory reaction and the firm adhesion that monocytic series THP-1 produces greatly under emulation physiology flow regime.Thus, the expression of fat-1 provides a kind of potential methods of treatment for the treatment of similar atherosclerotic inflammatory vascular disease.

Embodiment 11: as the n-3 dehydrase of antiarrhythmic

For whether the expression of judging fat-1 can provide antiarrhythmic effect, whether the myocyte that then need detect expression n-3 dehydrase is to causing the heart murmur symptom sensitivity that heart murmur reagent causes.Make newborn mouse cardiac myocyte infect Ad.GFP.fat-1 or Ad.GFP, wherein this myocyte cultivates on cover glass and can spontaneously beat.Infect two days later, to filling system, it is full of the serum-free medium that contains high concentration (5-10mM) calcium ion with these cell transfer.These culture fluids are to cause heart murmur reagent.In this filling process, use phase contrast microscope and video detector to detect the myocyte and shrink situation.When high-calcium ionic concentration reaches 7.5mM, infect that the cellular control unit that Ad.GFP is arranged takes place at once that spasm is shunk or fibrillation after show jumping frequency rate rapidly and accelerate, can keep normally and beat and infect the cell that Ad.GFP.fat-1 is arranged.Thus, the myocyte who expresses the n-3 dehydrase stimulus that caused heart murmur hardly influences (seeing also Figure 13).

Embodiment 12:fat-1 expresses and the inhibition tumor growth

For test cdna shifts effect to tumor growth in vivo, we do experiment on two nude mices that suffer from human breast cancer xenograft (MDA-MB-231).To one of them mouse intratumor injection 50mlAd.GFP.fat-1 (1012 particles/ml) every three days.An other injection control group carrier (AD.GFP).Monitoring 4 week of tumor growth rate.Experiment shows that injection has the growth of tumor speed of Ad.GFP.fat-1 than control group tumour slow (seeing also Figure 14).

The expression of embodiment 13:fat-1 is to the growth of mankind mastopathy cell in the influence of aliphatic acid composition and the culture fluid

Make up recombined adhenovirus (Ad): make up the recombined adhenovirus (Ad) (please refer to Kanget al., Proc.Natl.Acad.Sci.USA 98:4050-4054,2001) that carries fat-1 cDNA gene according to aforesaid method.In brief, fat-1 cDNA insertion gene is sheared out and is inserted into the transfer vector through the dual cleavage reaction of limiting enzyme EcoRI/KpnI from plasmid among the pCE8 (as previously mentioned), carry out the recombined adhenovirus skeleton according to the method for He et al. (please refer to Proc.Natl.Acad.Sci.USA95:2509-2514,1998) then.Form two first generation 5 type recombined adhenovirus: carry the green fluorescent protein (GFP under the control of macrophage type virus (CMV) enhancer, as reporter gene) Ad.GFP, and carry fat-1 gene and GFP gene and the Ad.GFP.fat-1 under cmv enhancer control separately.As mentioned above, recombinant virus is bred in 293 cells and is become the virus (please refer to Hajjaret al.Circulatin 95:423-429,1997) of high titre value.This structure is sheared by enzyme and dna sequence analysis is determined.

Cell culture and adenovirus infection: the MCF-7 cell according to routine remain on the DMEM that mixes with 1: 1 (volume ratio) and Ham ' s F12 culture fluid (JRH, Bioscience) in, wherein contain 5% hyclone (FBS) and antibody-solutions (50U/ml penicillin; 50 μ g/ml streptomycins), temperature is controlled at 37 ℃, and places the tissue culture incubator (to contain 5%CO ₂With 98% to humidity) in.Party adds virion (3-5*10 to serum-free medium to 70% the time when cell enlargement ⁸Particles/ml), in order to do making cell infection adenovirus Ad.Originally, best virus concentration is by deciding by using Ad.GFP to make viral toxic minimize with the optimum balance that obtains high gene and express and hang down virus titer.After cultivating in 24 hours, it is alternative with the conventional culture fluid of 18:2n-6 that is supplemented with 10 μ M and 20:4n-6 to infect culture fluid.Infect after 48 hours, this cell is used for analyzing gene expression, fatty acid composition, eicosanoid product and cell proliferation and cell death.

RNA analyzes: the Fat-1 gene expression dose can be by using RPA III ^TM(provided by Ambion company, Austin TX) adopts RNase enzyme protection test method to measure to equipment.In brief, the step of drafting according to manufacturer use total RNA separation agent (TRIzol, GIBco BRL) with total R NA from through cultured cells, extracting.Like this, the plasmid pCE8 that has the fat-1 gene is linearized, and can be used as and transcribe template.Use ³³P-UTP, T7 polymerase (Riboprobe System ^TMThe T7 instrument is provided by Promega) transcribe antisense RNA probes in the body, this antisense RNA probes through extracting from the cardiac muscle cell total RNA (15 μ g) hybridization and shear in order to do the RNA and the probe that will not have to hybridize are removed with the RNase enzyme.Shielded RNA-RNA is dissolved in the sex change sequence gel, and carries out video picture according to autoradiography.The probe of the GAPDH gene that points to is as the internal contrast group.

Because the infected cell and the GFP coexpression (demonstrating bright fluorescence) of express transgenic so just are easy to identify these cells by fluorescence microscope.Infect after 3 days, the cell of observing 60-70% is infected and by transgene expression.Use RNase enzyme protection test method to analyze mRNA and show, in infection has the cell of Ad.GFP.fat-1, profuse fat-1mRNA is arranged, and in infection has the cell of Ad.GFP (control group), do not detect fat-1 mRNA.This result shows, is originally lacking in the mouse cortex nerve cell of fat-1 gene, and the gene transfer that adenovirus Ad gets involved the back generation brings the height of fat-1 gene to express.

Lipid analysis: be the efficient of check gene transfer in the fatty acid of the human MCF-7 cell of modification is formed, extract total bioblast and adopt gas chromatography to analyze at infection adenovirus Ads and after n-6 fatty acid is cultivated 2-3 days.(please refer to Kang et al., supra) fatty acid is formed in the analyzing total bioblast as previously mentioned.With the chloroform/methanol that contains 0.005% butylated hydroxytoluene (as age resistor) (2: 1, volume ratio) solution lipid is extracted.Adopt 14% (wt/vol) BF ₃/ methanol reagent prepares fatty acid methyl ester.Utilization gas chromatography/mass spectrometry technology is measured the amount of fatty acid methyl ester, and used experimental instrument is HP5890 Series II gas chromatograph and SupelcowaxSP-10 polarity capillary chromatographic column (Supelco, Bellefonte, HP5971 mass spectrograph PA) are housed.Syringe and detector respectively remain on 260 ℃ and 280 ℃.Baking oven program originally temperature is 150 ℃, kept 2 minutes, rises to 200 ℃, keeps 4 minutes with 10 ℃/min speed then, rises to 240 ℃, keeps 3 minutes with 5 ℃/min speed again, rises to 270 ℃, keeps 5 minutes with 10 ℃/min speed at last.Carry gas flow rate to remain 0.8mL/min.In molecular weight range 50-550AMUs, carry out total ion monitoring.By the relatively more different fatty acid zones of analyzing and internal standard fixed concentration zone, measure the fatty acid molecule mass number.

The expression of fat-1 cDNA makes n-6 fatty acid be converted into n-3 fatty acid in the MCF-7 cell, thus n-6: the ratio generation great change of n-3 fatty acid.The fatty acid spectrogram that obtains with reference to cell and infect spectrogram that the Ad-GFP-fat-1 cell is arranged between a great difference (seeing also Figure 15) is arranged.When infection had the cell of Ad-GFP to compare with the cell that does not infect, fatty acid composition did not change.And have in the cell of fat-1 cDNA (n-3 dehydrase) in expression, various n-6 fatty acid all are converted into corresponding n-3 fatty acid, and for example, 18:2n-6 is converted into that 18:3n-3,20:4n-6 are converted into 20:5n-3,22:4n-6 is converted into 22:5n-3.Therefore, party expresses in the cell of fat-1 gene fatty acid and forms and infect in the cellular control unit that Ad.GFP is arranged the fatty acid ratio of components when (seeing also Figure 15), and the ratio of finding n-6: n-3 12 is reduced to 0.8 in the cell of expressing the n-3 dehydrase in cellular control unit.

Measure eicosanoid: the front shows, is derived from that the wherein a kind of main eicosanoid PGE2 (PGE2) of 20:4n-6 (arachidonic acid) precursor is relevant with tumor growth (to please refer to Rose and Connolly, Pharmacol.Ther. 83: 217-244,1999; Cave, BreastCancer Res.Treat. 46: 239-246,1997).Whether can bring the difference of eicosanoid product in the cell for the difference of judging arachidonic acid and arachic acid content, then need measure PGE2 product in the infected cell, the enzyme immune detection instrument that PGE2 needs to be derived from the PGE2 of AA by special detection before measuring starts with calcium ion channel A23187, and wherein PGE2 and PGE3 have 16% cross reaction.Know clearly it, use enzyme immune detection instrument (providing) to measure PGE2 by Assay Designs company according to the step that manufacturer is drafted.(16% cross reaction being arranged) with PGE3.Clean the cell of cultivating with the PBS buffer solution that contains 1%BSA, then this cell is put into the same buffer that contains calcium ion channel A23187 (5 μ M) and cultivated.Cultivate after 10 minutes, recover this adjustment medium, be used to measure eicosanoid then.The amount of the PGE2 that is produced by the fat-1 cell reduces (seeing also Figure 16) greatly than the amount of the PGE2 that is produced by cellular control unit.

Analysis of cells breeding and cell death:, need after gene transfer, to measure cell proliferation and cell death situation for judging of the effect of fat-1 expression of gene to the growth of MCF-7 cell.According to conventional method, utilize microscope detect cellular morphology (Si Wang cell through single from, round and little), and can judge cell quantity total in every well by the method for using the hemacytometer calculating survivaling cell.In addition, also available MTT cell proliferation equipment (being provided by Roche Diagnostic Corporation) is measured the cell proliferation ability.The step that dead cell can be drafted according to manufacturer is used to dye to be had VybrantTMApoptosis Kit #5 the atomic nucleus of (molecular probe Molecule Probes) is judged.

Metamorphosis (round and little cell or fragment) and atomic nucleus dyeing (sapphirine) show that the cell of great expression fat-1 gene suffers death.The statistical results show of dead cell quantity infects that 30-50% is arranged in the cell that Ad.GFP.fat-1 is arranged is dead cell, and only finds 10% dead cell in cellular control unit (infection has Ad.GFP).MTT the analysis showed that the fertility that infects the cell that Ad.GFP.fat-1 is arranged has the cell of Ad.GFP much lower than infecting.Like this, infect the heavy survivaling cell sum of cell that Ad.GFP.fat-1 is arranged than approximately lacking 30% in the cellular control unit.These results are consistent with this proposal of fat-1 expression useful asticancer agents.

Data analysis, statistical analysis: the data of appearance are mean value ± SE.Student ' T test is used to assess the difference of two numerical value.The horizontal p of validity＜0.05.

Embodiment 14: the cultivation of transgenosis fish

The gene of coding n-3 fatty acid dehydrogenase (no matter be wild type gene, the gene optimized, or the bioactive fragment of other tool or its variant) can be used to cultivate the transgenosis fish that have modification n-6 fatty acid.Can use structure that traditional gene transfer method will contain dehydrogenase gene and enhancer to construct body and transfer in the fish animal, for example be used for zebra fish (adopting the sperm nucleus trasplanting method), as Jesuthasan et al. (Dev.Biol. 242: 88-95.3,2002) described.This Jesuthasan method will be described below.

Preparation sperm nucleus: the interior testis that downcuts of bull zebra fish (Daniorerio) body that places frozen water certainly.The testis that will be in the bladder both sides with tiny tweezers takes out.After Kroll and the described method of Amaya (Development 122:3173-3183,1996) done some modifications (as saving protease inhibitors), preparation striping sperm nucleus.Use lysolecithin (Sigma L4129) or digitophyllin (Sigma D5628) striping.For ease of checking the concentration after cleaning, sperm nucleus is sticked Hoechst or Syto11 (molecular probe) label, and count with hemacytometer.The 10 mul aliquots samples that concentration is approximately 100nuclei/nl quick-frozen and being stored under-80 ℃ in liquid nitrogen.Also can use another kind of sperm nucleus is method by the striping that thaws: the sperm nucleus that sperm nucleus contains 5%BSA at 9ml is isolated in the culture fluid and is cleaned twice; in 1ml, clean once again, and finally before by five equilibrium, be suspended in 250 microlitres again and IQF under the situation of not freezing protection.

Sneak into transgenosis: plasmid DNA is purified through Qiaquick post (Qiagen) through the suitably enzyme material linearisation of restriction, is diluted to concentration 70ng/ μ l with sterile water then.The sperm aliquot is thawed, then with the most advanced and sophisticated pipette through excision of white by on move down liquid mode mix.5 microlitre sperm suspensions are transferred in the Eppendorf test tube of 1.5ml, add 1 μ l linear DNA then.If need to use a large amount of DNA in the experiment, can improve storage concentration, but the volume that is added must remain on 1 μ l.Employing moves the liquid mode and sneaks into, and at room temperature left standstill 1 minute, 5 minutes, 20 minutes, use following sperm dilution buffer liquid (sperm dilute buffer then, SDB) dilution: 250mM sucrose solution, 75mM KCl solution, 0.5mM three hydrochloric acid spermidine (spermidine trihydrochloride) solution, the smart ammonia solution of 0.2mM four hydrochloric acid, pH 7.4, or MOH buffer solution (10mM KPO ₄Solution, pH7.2,125mM gluconic acid potassium solution, 5mM Nacl solution, 0.5mMMgCl ₂Solution, 250mM sucrose solution, 0.25mM three hydrochloric acid spermidine solution, the smart ammonia solution of 0.125mM four hydrochloric acid), finally be diluted to 500 μ l.Before using, this diluted mixture liquid is placed on the ice cube.

Inject sperm nucleus: anaesthetize female zebra fish with tricaine (SigmaA 5040), accompany in Ti Shi (Petri) culture dish light then the stone roller to get rid of mature egg with a clean film inclosure of sealing.Ovum is put into a rickle, culture dish is in prevents desiccation immediately.With pasteurellaceae (Pasteur) pipette ovum (once about 50) is moved in the flood chamber, this flood chamber is made up of the V-type groove in 1.2% agarose that is formed at Hanks ' the s salt that contains 0.5%BSA, to delay activation.Fill this V-type groove, up to just the submergence ovum---the distance between upper end ovum and the lower end meniscus is no more than 1mm.This has just guaranteed that the injection pin effectively extracts out from ovum, and ovum is caught hold of by the surface tension of salting liquid.

Extract the thin-walled capillary from the pipette extractor, smash the tip with tweezers then and make outlet diameter, so just make the injection pin 10-15 μ m (diameter of sperm nucleus is approximately 5 μ m).Should inject pin and be installed on the support, be connected, and use micro-syringe to fill then from the tip with mechanical modulator.Because sperm nucleus is visible under dark field illumination, therefore can utilize disecting microscope to observe the filling situation.With 20 μ l pipettes the mixing material back-filling is imported in the tygon flexible pipe that is connected with yellow tip.Then this flexible pipe is connected with capillary, sperm suspensions is clamp-oned in this capillary, and be pressed onto most advanced and sophisticated place with 200 μ l pipettes.

Inject with the syringe that is set to pressure 3-5psi, time 100ms.Sperm nucleus is injected ovum animal polar region.Make ovum pass about 50-100 μ m from micropyle, this process is observable under the illumination of the bright visual field, rotates ovum then so that most advanced and sophisticated near micropyle in the preceding order of injecting.After injecting one group of ovum, with the Pasteur pipette they are moved to and to have 20ml E3 culture fluid (5mM NaCl, 0.17mM KCl, 0.33mM CaCl ₂, 0.33mMMgSO ₄) in the 9-cm Petri culture dish, place 28 ℃ of incubators then.Some development of fertilized ova are grown up, and intersection is just cultivated the offspring who expresses n-3 fatty acid after implanting the wild type fish.

According to the analytical method of trangenic mice, respectively genotype and phenotype are analyzed with RT-PCR and gas chromatography (lipid analysis).

See also Figure 20, be the wild type representing the fat-1 gene to be arranged from expression with the part gas chromatogram with transgenosis type zebra fish muscular tissue the different polyunsaturated fatty acids spectrogram of the TL that extracts, wherein the method for modifying of fat-1 gene is as described in example 8 above.The contents level of omega-3 fatty acid is much higher than the wild type fish in the tissue of transgenosis fish, and the contents level of omega-6 fatty acid is significantly less than the wild type fish.Construct with identical described in the embodiment 8 as the DNA that cultivates transgenosis fish animal.

Also can use other method that foreign gene is implanted in the zebra fingerling system.These methods comprise that plasmid DNA is injected the method for body early embryo, the gene insertion of transposons intervention or the gene transfer method that retrovirus is got involved (please refer to Udvadia AJ and Linney E.Windows into development:historic, current, and future perspectives on transgeniczebrafish, Development Biology, 2003 256: 1-17; Detrich, H.I., Westerfield, M., Zon, L.I. (Eds): Thezebrafish.In Methods in Cell Biology 1999:Vol.59 ﹠amp; 60).

Method described here also can be applicable to comprise other bony fish of salmon (as Atlantic Ocean salmon, Atlantic salmon).

Other additional information can supply to know personage's reference of common skill in the present technique field: Simopoulos and Cleland, World Rev.Nutr.Diet (Basel, Karger) Vol.92,2003; Simopoulos et al. (Eds) .World.Rev.Nutr.Diet. (Basel, Karger) Vol.83,1998; Connor, Am.J.Clin.Nutr. 71: 171S-175S, 2000, Simopoulos, Am.J.Clin.Nutr. 70: 560S-569S, 1999; Salem et al., Lipids 31: S1-S326,1996; Leaf and Weber, Am.J.Clin.Nutr. 45: 1048-1053,1987).

Embodiment 15: cultivate transgene pig

Use the method for similar cultivation trangenic mice recited above to cultivate transgene pig.Transgene pig is expressed the fat-1 gene, and the method for modifying of this gene as described in example 8 above.Seeing also Figure 21, is the different polyunsaturated fatty acids spectrogram of representing to have from expression the TL that extracts the afterbody tissue of the wild type of fat-1 gene and transgenosis type pig with the part gas chromatogram.The contents level of omega-3 fatty acid is much higher than the wild type pig in the tissue of transgene pig, and the contents level of omega-6 fatty acid is significantly less than the wild type pig.Construct with identical described in the embodiment 8 as the DNA that cultivates transgene pig.

So far a large amount of embodiment of explanation embodiment of the present invention have all been described.Yet under not departing from spirit of the present invention, can carry out various modifications and change.

Claims (45)

1. One kind through single from nucleic acid molecules, it comprises: a sequence, the enzyme material of this sequential coding can become the dehydrogenation of n-6 fatty acid in corresponding n-3 fatty acid, wherein this sequence comprises at least one optimizing codon.
2. 2. as claimed in claim 1 through single from nucleic acid molecules, wherein this sequence is a C.elegans fat-1 gene.
3. 3. as claimed in claim 2 through single from nucleic acid molecules, wherein this sequence comprises at least 5, maximum 150 optimizing codon.
4. 4. as claimed in claim 2 through single from nucleic acid molecules, wherein the 5-10 of this codon, 10-15,15-20,20-25,25-30,30-35,35-40,40-45,45-50,50-55,55-60,60-65,65-70,70-75,75-80,80-85,85-90,90-95,95-100,100-105,105-110,110-115,115-120,120-125,125-130,130-135,135-140,140-145 or 145-150 are optimised.
5. 5. as claimed in claim 1 through single from nucleic acid molecules, it comprises and is positioned at position 6,9,18,20,22,24,28-30,33-36,47,49,52,54,58,60,61,64,67,69-71,73,77,79,81,86,89,92,94-95,100,101,105,106,112,115,118,124,127,128,131,146,151,154,161,163,164,169,178,187,188,195,197,200,202,206,210,214,217,221,223,225,227,228,232,234,241,245,255,271,280-282,284,285,301,303,310,312,327,362 or 370 optimizing codon.
6. 6. as claimed in claim 2 through single from nucleic acid molecules, it comprises nucleotide sequence as shown in figure 18.
7. 7. as claimed in claim 1 through single from nucleic acid molecules, comprise the nucleotide sequence of the therapeutical peptide of encoding again.
8. 8. as claimed in claim 1 through single from nucleic acid molecules, comprise the adjusting part or the nucleotide sequence of code identification again.
9. 9. as claimed in claim 8 through single from nucleic acid molecules, wherein this adjusting part is tissue-specific promotor.
10. 10. expression vector, it comprises the described nucleotide sequence of claim 1.
11. 11. expression vector as claimed in claim 10, wherein this carrier is a viral vectors.
12. 12. expression vector as claimed in claim 11, wherein this viral vectors is retroviral vector or adenovirus vector.
13. 13. expression vector as claimed in claim 10, wherein this carrier is a plasmid.
14. 14. a host cell, it comprises the described nucleic acid molecules of claim 1.
15. 15. a host cell, it comprises the described expression vector of claim 10.
16. 16. a medical composition, it comprises described expression vector of claim 10 and physiologically acceptable dilution.
17. 17. a non-human transgenic animal, it comprises the described nucleic acid molecules of claim 1.
18. 18. non-human transgenic animal as claimed in claim 17, wherein this animal is mammality, birds or fish.
19. 19. non-human transgenic animal as claimed in claim 18, wherein this mammality is ox, pig or sheep; These birds are chicken, turkey, duck, goose or pheasant; And these fish are salmon, trout or yaito tuna.
20. 20. the food or the product of extra-nutrition, it comprises described this non-human transgenic animal of claim 17 or its tissue or its part through processing.
21. 21. comprising to this individuality, a method that improves n-3 content of fatty acid in the individual diet, this method throw the described food of claim 20 or the product of extra-nutrition of giving.
22. 22. a treatment suffers from the patient's of cancer method after diagnosing, this method comprises to this patient throws the described nucleic acid molecules of claim 1 that gives the treatment effective dose.
23. 23. method as claimed in claim 22, wherein this cancer is breast cancer, colon cancer, prostate cancer, liver cancer, cervix cancer, lung cancer, the cancer of the brain, cutaneum carcinoma, cancer of the stomach, Head and Neck cancer, cancer of pancreas, leukemia or oophoroma.
24. 24. comprising to this individuality, a method that suppresses nerve cell death in the individual body, this method throw the described nucleic acid molecules of claim 1 that gives the treatment effective dose.
25. 25. method as claimed in claim 24 wherein should individuality be diagnosed as and have suffered from neurodegenerative disease.
26. 26. method as claimed in claim 25, wherein this neurodegenerative disease is Alzheimer's (Alzheimer ' s disease), Parkinson's disease (Parkinson ' s disease) or Huntington (Huntington ' sdisease).
27. 27. treat individual method for one kind, this individuality suffers from maybe may develop the illness uneven relevant with the ratio that lacks n-3 polyunsaturated fatty acids (PUFA) or n-3:n-6 PUFA, and this method comprises to this individuality throwing gives the described nucleic acid molecules of claim 1.
28. 28. method as claimed in claim 27, wherein this illness is cardiac arrhythmia, angiocardiopathy, cancer, inflammation (for example, such as atherosclerotic inflammatory vascular disease or such as the vascular disorder of ISR), autoimmune disease, retina or brain deformity or tendency deformity, diabetes, obesity, cutaneous disorder, ephrosis, ulcerative colitis, Crohn's disease (Crohn ' sdisease) or COPD.
29. 29. treat individual method for one kind, the graft of biologic-organ, tissue or cell has been accepted or prepared to accept to comprise to this individuality, this method comprises to this individuality or the throwing of this graft gives the described nucleic acid molecules of claim 1.
30. 30. transgenosis fish, it comprises a nucleotide sequence, and the enzyme material of this sequential coding can become the dehydrogenation of n-6 fatty acid corresponding n-3 fatty acid.
31. 31. transgenosis fish as claimed in claim 30, wherein these fish are cod or any gadidae, Gadiformes fish; Halibut; Catfish or any Pacific herring order fish; Mackerel or any mackerel section fish; Salmon or any salmon fishes comprise trout; Perch or any bacalao; Westerly wind or any clupeidae fish; Ray or any Rajidae fish; Flatfish or any Osmeridae fish; Sole or any Soleidae fish; And yaito tuna or any mackerel section fish.
32. 32. transgenosis fish as claimed in claim 30, wherein this nucleotide sequence comprises C.elegans fat-1 gene.
33. 33. transgenosis fish animal as claimed in claim 32, wherein this C.elegans fat-1 gene comprises at least one optimizing codon.
34. 34. transgenosis fish animal as claimed in claim 33, wherein this sequence comprises at least 5, maximum 150 optimizing codon.
35. 35. transgenosis fish animal as claimed in claim 33, wherein the 5-10 of this codon, 10-15,15-20,20-25,25-30,30-35,35-40,40-45,45-50,50-55,55-60,60-65,65-70,70-75,75-80,80-85,85-90,90-95,95-100,100-105,105-110,110-115,115-120,120-125,125-130,130-135,135-140,140-145 or 145-150 are optimised.
36. 36. transgenosis fish animal as claimed in claim 33, wherein this nucleic acid comprises and is positioned at position 6,9,18,20,22,24,28-30,33-36,47,49,52,54,58,60,61,64,67,69-71,73,77,79,81,86,89,92,94-95,100,101,105,106,112,115,118,124,127,128,131,146,151,154,161,163,164,169,178,187,188,195,197,200,202,206,210,214,217,221,223,225,227,228,232,234,241,245,255,271,280-282,284,285,301,303,310,312,327,362 or 370 optimizing codon.
37. 37. transgenosis fish as claimed in claim 33, wherein this nucleic acid comprises nucleotide sequence as shown in figure 18.
38. 38. a transgenic bird, it comprises a nucleotide sequence, and the enzyme material of this sequential coding can become the dehydrogenation of n-6 fatty acid in corresponding n-3 fatty acid, and wherein these birds are fed for consumption.
39. 39. transgenic bird as claimed in claim 38, wherein these birds are chicken, turkey, duck, goose or pheasant.
40. 40. transgenic bird as claimed in claim 38, wherein this nucleotide sequence comprises C.elegans fat-1 gene.
41. 41. transgenic bird as claimed in claim 40, wherein this C.elegans fat-1 gene comprises at least one optimizing codon.
42. 42. transgenic bird as claimed in claim 41, wherein this C.elegans fat-1 gene comprises at least 5, maximum 150 optimizing codon.
43. 43. transgenic bird as claimed in claim 42, wherein the 5-10 of this codon, 10-15,15-20,20-25,25-30,30-35,35-40,40-45,45-50,50-55,55-60,60-65,65-70,70-75,75-80,80-85,85-90,90-95,95-100,100-105,105-110,110-115,115-120,120-125,125-130,130-135,135-140,140-145 or 145-150 are optimised.
44. 44. as the transgenic bird of claim 41, wherein this C.elegansfat-1 gene comprises and is positioned at position 6,9,18,20,22,24,28-30,33-36,47,49,52,54,58,60,61,64,67,69-71,73,77,79,81,86,89,92,94-95,100,101,105,106,112,115,118,124,127,128,131,146,151,154,161,163,164,169,178,187,188,195,197,200,202,206,210,214,217,221,223,225,227,228,232,234,241,245,255,271,280-282,284,285,301,303,310,312,327,362 or 370 optimizing codon.
45. 45. as the transgenic bird of claim 41, wherein this C.elegansfat-1 gene comprises sequence as shown in figure 18.

Images