CN101532018A - Producing method of transgenic mouse containing anthropogenic proto-oncogene c-Ha-ras and application thereof - Google Patents
Producing method of transgenic mouse containing anthropogenic proto-oncogene c-Ha-ras and application thereof Download PDFInfo
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Abstract
The invention provides a producing method of a transgenic mouse integrated with anthropogenic proto-oncogene c-Ha-ras DNA in genomes. DNA transferred contains four coding exons, introns and regulatory sequences of human c-Ha-ras gene. The regulatory sequences are preferentially selected from the promoter region of the human c-Ha-ras gene and a poly A tailing signal sequence. The transgenic mouse, filial generation, embryo, organ and/ or derived cells produced with the method can be used for canceration mechanism study, carcinogen or cancer inhibitor screening research and pre-clinical safety evaluation of drugs.
Description
Technical field
The present invention relates to make the method for cancer transgenic animal model; The purposes of this animal model during safety evaluation is studied at the canceration mechanism research of multiple cancer, carcinogens or before pressing down screening study, medicine clinical of cancer thing; And cell, tissue, organ and embryo and the purposes in above-mentioned field thereof of deriving out by this animal pattern.
Background technology
Since the beginning of the eighties, the first transgenic mice was born, set up and developed more method of gene introduction, the transgenosis, embryonic stem cell technology, nuclear transfer technology, gene knockout (knocking in) technology and the RNA that comprise microinjection, retrovirus mediation interfere, and sperm vector etc., and numerous patents (United States Patent (USP): US6576811B1,2003) have also been produced thus.
Wherein, because its stability and success ratio are higher, micro-injection method becomes one of genetically modified method the most commonly used.It is that the solution direct injection that will contain recombinant DNA enters among the embryo, as a rule, is that DNA is expelled in the male pronucleus.When being used for the dna solution of microinjection, generally adopt enzyme to cut, reclaim target DNA fragment refining, steps such as gel DNA purifying obtain purified DNA sample.Suitable microinjection pin and hold that to decide pin be the key that DNA successfully is injected into embryo's male pronucleus of 0.5 day.Embryo's quality also is the key of transgenosis success, and good embryony rule is full.
Being a by force technical but very direct surgical technic through the embryo transfer of microinjection to the female mouse acceptor of false pregnancy, its complete set operation must be accomplished aseptic, quick and dexterous.After treating the conceived cub of false pregnancy mouse, by clip young baby tail point or to obtain the part ear by punch tool wealthy, the possible transgenosis head that obtained for 2～4 ages in week builds the tissue sample of mouse, is used to extract DNA, determines the integration situation of goal gene by PCR or Southern hybridization technique.
Mouse can be used for mating and build and be when 6～8 all rheological properties are ripe.The transgenic progeny of birth is also identified by PCR or Southern hybridizing method, retains and contains genetically modified animal, and further breeding enlarges, and 1. observes genetically modified expression and heredity by breeding of several generations so that realize; 2. enlarge colony's quantity and provide more mouse for experimental study; And 3. produce the transgenic mouse and compare of isozygotying with the heterozygosis transgenic mouse.
The foundation of disease animal model is the key areas of transgenic research, and wherein the foundation of animal model for cancer particularly comes into one's own.This is because cancer is the second largest disease that threatens mankind nowadays life.According to WHO, there were 5,800 ten thousand people's Died Of Diseases in the whole world in 2005, and two wherein have 13% for suffering from cancer mortality.Development (Wynn, 2007 that a large amount of funds and manpower are used for research of cancer genesis mechanism and curative drug are constantly dropped in countries in the world; Clin Invest.117:524-529; Galanski and Keppler, 2007, Anticancer Agents Med Chem, 7:55-73).But, no matter be in the fundamental research field or in the medicament research and development field, the capital runs into for want of suitable evaluation means and research platform and causes the passive situation of work stagnation, for adapting to this demand, the various genetic modification animal models of the receptor genome being transformed by the modern biotechnology means (Genetic Modified Animal model) arise at the historic moment (Barone Metal, 2006, Mol Med, 12:115-23; Choedon et al, 2006, World JGastroenterol, 12:2517-2522).The promotion that the appearance of cancer transgene mouse model is strong the development of cancer research, it is to carry out relevant oncogenesis, development, transfer (Tanaka et al in molecular level and integral level, 2006, Ann N YAcad Sci.2006,1086:1-10) and study on prevention (Galanski and Keppler, 2007, Anticancer Agents Med Chem, good living materials 7:55-73), also be the good platform (Mitsummori of the development of cancer related drugs, new drug safety evaluation, 2003, J Toxicol Sci.28:371-383).Therefrom the dependency of information of Huo Deing and data and these data and human body is that other stripped means are difficult to replace.
For the carinogenicity evaluation, the experiment of traditional carinogenicity adopts big mouse to carry out, and time long (needing 2 years), expense height, specific aim is poor as a result.Given this, international bodies such as ICH advocate with leading the carinogenicity that new means are estimated medicine, and the transgenosis animal model for cancer is desirable alternative means.Therefore, it is very necessary to set up outstanding animal model for cancer.
RasH2 transgenic mice and rat (U.S. Pat 6576811B1) are exactly one of this type of cancer transgenic models.This mouse model was set up (Katsuki et al, 1989) by Japanese Experimental Animal Center institute at first in 1989, importing be people's normal total length ras gene (c-Ha-ras) (Sekiya et al, 1985).C-Ha-ras is a member of ras proto-oncogene family, the p21 albumen that coding is made up of 189 amino acid.P21 albumen is the signal of interest transduced element in the signal transducting system in the cell, and the propagation of pair cell and differentiation play regulating effect (Douglas R, 1993).The 12nd, 13,61 codings of ras gene sudden change can causing p21 protein conformation changes, and causes taking place multiple cancer (Mitsummori, 2003).The rasH2 cancer model is having crucial purposes aspect the carinogenicity risk assessment.According to International Life Sciences Institute (ILSI) in 2003, environment and health science institute (HESI) and international toxicity project how tame international bodies such as (NTP) are to the joint test of tens kinds of known genotoxic carcinogenses, find that this model can distinguish carcinogens and non-carcinogenic thing accurately, be quick on the draw, reliable results, only needed for 26 weeks experimental period, is to meet the best alternative model (substituting the big mouse carinogenicity experiment of traditional 2 terms) that ICH new drug short-term carinogenicity detects governing principle.
Done a large amount of work already aspect the carcinogenic mechanism research inducing, deepened the understanding (Mitsummori, 2003) of people mechanism of carcinogenesis.Originally it is believed that, change multiple copied people proto-oncogene over to after, for chemical carcinogen with proto-oncogene be converted into oncogene provide more target spots (Saitoh et al, 1990, Oncogene 5:1195-1200), causes this model more responsive to carcinogens.Transgenosis point mutation activation also is a reason, as generation (Ando et al, 1992, Cancer Res, the 52:978-982 of glandular stomach and cutaneous tag; Mori et al, 2000, Toxicol.Pathol.13:165-172); But for liver tumor, with the dependency of point mutation unclear (Hayashi et al, 1998, Toxicol.Pathol.26:556-561).In studies show that afterwards, the transgene product of cross expressing and canceration signal path other oncogene and cancer suppressor gene interaction may be this model to the underlying causes of carcinogens sensitivity (Tamaoki, 2001, ToxicolPathol.29suppl:81-89).Induce down at carcinogens, cks1b is raised in the endogenous ras genetic expression of mouse, tpm1, reck, the fact (OkamuraM that cancer associated gene transcriptional levels such as gelsolin change, et al, 2007, Cancer Lett.245:321-330) provides half-proof for this hypothesis.
However, since the gene source of this model in a tumour patient (Sekiya T et al, 1984; 1985), originally undergo mutation.In order to obtain normal transgenosis, have to through the operation of multistep molecular biology, correcting mutant site.On the other hand, the transgenosis of this model is bigger, at the 5-end long " redundancy " sequence is arranged.These are not enough can to increase difficulty to genetic manipulation, and is unfavorable for repetition.
Given this, the applicant has made up the transgenic animal model that contains people's proto-oncogene c-Ha-ras in real work.The transgenosis of this model comes from normal people's genome, can be directly used in transgenosis after separation is finished.Transgenosis is optimized, reduced 0.4kb at the 5-end than previously used transgenosis.
What is more important, the animal model of acquisition has original phenotype.Transgenosis in the past belongs to the induction type model, its outward appearance and body weight and normal mice almost are as good as (http://www.rash2.com/-phenotype), generally only when giving positive carcinogens, just induce the generation tumour, the probability of spontaneous knurl very low (＜1.1%), time of origin (8～27 weeks in evening, the skin squamous-cell papilloma, http://www.rash2.com/-background data).And the head that this research obtains built about mouse birth 2 weeks of back, i.e. the visible knob of spontaneous generation at the back, and fast growth, and develop into many places by a place.That is to say, compare with the rasH2 model of Japan that the cancer transgenic models that this research obtains has become spontaneous type by induction type, the time of generation shows to can be used as a new animal model for cancer than early, is used for the research of cancer genesis mechanism.Mainly show: this model is a spontaneous type, and time of origin is more Zao than Japanese rasH2 model, and this has hinted that this model may be more more responsive than rasH2 for carcinogenic reaction, is more suitable for being used for the research of cancer genesis mechanism aspect.The underlying causes that takes place in view of human cancer is " human body itself and the long-term results of interaction of environmental factors ", and generally speaking, it is very low that human body contacts the strong carcinogenic probability of similar heavy dose, so have reason to believe, it is truer near human body that the data ratio of the relevant cancer mechanism that the spontaneous type model draws brings out type.In addition, this model is the good material of transgenosis Phenomenological Research.There are some researches show that genetically modified expression intensive is subjected to the influence of host chromosome integration site, the height of transgene expression level and the relation of copy number be star very not, and relevant with integration site.Obtain transgenosis head in this research and build mouse and promptly occurred knob in early days, hinted that genetically modified expression level is high or undergo mutation (Xi Tao, 2001, the medicine biotechnology, 2001,8:301-305).This model compares research with the model of Japan, can answer the basic problems such as relation of inserting site and transgene expression.Therefore, aspect fundamental research, this model also will have important purposes.
The spontaneity of tumour and host's genetic background have bigger relation.It is disadvantageous that spontaneous tumor is used in safety evaluation animal pattern, because spontaneous tumor can induce the tumour of generation to obscure with the thing to be tried that the carinogenicity risk is arranged.In order to address this problem, can hybridize with transgenic animal C57 and another inbred lines such as BALB/c, use the F1 that produces to get final product.
Summary of the invention
The present invention relates to contain the making method and uses thereof of the transgenic mice of anthropogenic proto-oncogene c-Ha-ras.This animal model is by the artificial anthropogenic proto-oncogene c-Ha-ras that changed over to.This gene is from human normal tissue, and transgenosis contains the regulating and controlling sequence of this gene, whole exon and intron, with the expressed proteic integrity of abundant assurance.The approach of implementing this invention mainly contains microinjection, is one embodiment of the present invention.At first isolated genes from tissue obtains transgenosis through multistage purification refine, injects the mouse embryo through micro-injection method, is transplanted to then in the false pregnancy mouse body; Detect through molecular biology method, obtain to contain genetically modified positive transgenic animal; Further mating is built and is, obtains the transgenic animal population.On this basis, can further obtain cell, organ and the embryo of this animal model.
People's proto-oncogene c-Ha-ras of the present invention, its sequence is shown in SEQ ID NO:1.Described people's proto-oncogene c-Ha-ras contains 4 codings exon, promoter region and polyadenylic acid tailing signal sequence polyA.Wherein said promotor is the promotor of rodents villin promotor, cytomegalovirus CMV; Describedly add the polyA signal sequence that tailer sequence is SV40, BGH.
The present invention also comprises the host cell that contains described people's proto-oncogene c-Ha-ras, and the tissue that is formed by described host cell, organ.
The present invention relates to described host cell, tissue or organ in carcinogens screening or estimate, press down purposes in the safety evaluation of screening, medicine of cancer thing; Purposes in toxicology and the purposes in immunotoxicology.
The present invention relates to prepare the method for the rodents zygote of containing described people's proto-oncogene c-Ha-ras in the genome, the steps include:
(1) separates acquisition people proto-oncogene c-Ha-ras gene;
(2) obtain rodents zygote by super ovulation, mating, zygote screening and separating step;
(3) by micro-injection method the c-Ha-ras gene is injected zygote, obtain to contain the rodents zygote of people's proto-oncogene c-Ha-ras;
The invention still further relates to the method that contains the rodents transgenic animal of described people's proto-oncogene c-Ha-ras in the preparation genome, step is:
(1) makes the male mouse of vasoligation, allow itself and normal heat mouse mating then, obtain the false pregnancy mouse;
(2) be implanted in the uterine tube of false pregnancy mouse during incubated overnight to 2 cell with zygote directly transplanting as claimed in claim 9 or in the CO2 incubator;
(3) allow false pregnancy mouse cub, treat behind breast to detect by molecular biology method and obtain the transgenic positive filial generation, promptly head builds mouse;
(4) carrying out after head builds the mouse sexual maturity that mating builds is to obtain this transgenic animal.
Beneficial effect of the present invention is that the application Human To Human proto-oncogene c-Ha-ras optimizes, and makes it reduce 0.4kb than transgenosis of the prior art at the 5-end.What is more important, the animal model that provides method of the present invention to obtain has original phenotype, and the head that is obtained built about mouse birth 2 weeks of back, i.e. the visible knob of spontaneous generation at the back, fast growth, and develop into many places by a place.Be that the cancer transgenic models that the present invention obtains is a spontaneous type, the time of generation early shows to can be used as a new animal model for cancer, is used for the research of cancer genesis mechanism.
In addition, this model is the good material of transgenosis Phenomenological Research.There are some researches show that genetically modified expression intensive is subjected to the influence of host chromosome integration site, the height of transgene expression level and the relation of copy number be star very not, and relevant with integration site.Obtain transgenosis head in this research and build mouse and promptly occurred knob in early days, hinted that genetically modified expression level is high or undergo mutation (Xi Tao, 2001, the medicine biotechnology, 2001,8:301-305).This model compares research with the model of Japan, can answer the basic problems such as relation of inserting site and transgene expression.Therefore, aspect fundamental research, this model also will have important purposes.
The spontaneity of tumour and host's genetic background have bigger relation.It is disadvantageous that spontaneous tumor is used in safety evaluation animal pattern, because spontaneous tumor can induce the tumour of generation to obscure with the thing to be tried that the carinogenicity risk is arranged.In order to address this problem, can hybridize with transgenic animal C57 and another inbred lines such as BALB/c, use the F1 that produces to get final product.
Description of drawings
Fig. 1 show the used c-Ha-ras gene of the present invention structural representation and with prior art in the length contrast of (U.S. Pat 6576811B1) disclosed gene.The used transgenosis length of the present invention be 6.5kb (Fig. 1-A), contain 4 exons is followed successively by Ex1, Ex2, Ex3 and Ex4 represent with filled box; The disclosed transgenosis of prior art is 6.9kb (Fig. 1-B), have more 0.4kb in 5 ' the distolateral pterion than c-Ha-ras gene of the present invention.Shown among the figure with full gene splicing involved enzyme and cut the site.
Fig. 2 shows the complete sequence of transgenosis c-Ha-ras.Setting-out partly is 4 exon regions.
Fig. 3 shows the separation strategy of transgenosis c-Ha-ras.Isolation of genomic DNA from normal human blood adopts PCR method increase the respectively 1853bp in 5 ' end 1991bp, stage casing and the 3180bp of 3 ' end then.The 1853bp fragment 5 ' end of amplification contains the KpnI site, and at 3 ' end, adds the EcoRI site.Amplified fragments is cut with KpnI+EcoRI is two, obtains the fragment of 1400bp, inserts in same two pUC19 carrier of cutting, and obtains first intermediate carrier pUC19-1400.With the 1991bp fragment HindIII+KpnI of amplification, obtain to have the 1900bp fragment of two cohesive ends then, insert above-mentioned intermediate carrier pUC19-1400, obtain second intermediate carrier pUC19-3300.After amplification being obtained the sequencing fragment of 3180bp, downcut once more with EcoRI, and with pUC19-3300 EcoRI linearizing, and, obtain to contain total length transgenosis c-Ha-ras carrier pUC19-6500 with the insertion of 3180 fragments.Because 3180bp fragment two ends are the EcoRI site, need identify the direction of inserting, to obtain the correct carrier of direction of insertion.
Fig. 4 shows that head builds mouse many places knobs (shown in the arrow) promptly take place at the back in 2 weeks of birth.The antedorsal of Chu Xianing at first, circle, harder, about 2 * 3mm size, fast growth, 6 all left and right sides the maximum reach 1 * 1cm, and develop into many places by a place.This characteristic is former with not seeing description in the class model.
Fig. 5 shows that head builds the spontaneous tumor of mouse.In birth back about 1 year, there are two head to build mouse and remarkable tumour occur at neck, one remarkable tumour occurs at back part.Fig. 4-A tangible tumour occurs at neck; Fig. 4-B finds the many places tumour in lung, the greater figure wherein shown in the arrow.4-C separates tumour from the mouse health, and arrow is shown in big inside tumor and contains a plurality of small-sized tumours; Fig. 4-D, the length that display separation is got off has the lobe of the lung of tumour.Even when obvious tumour takes place these transgenic positive mouse, and the negative mouse phenotype of its brood transgenosis is normal.
Fig. 6 shows that genetically modified sudden change detects.Build mouse, comprise the tail point tissue extraction DNA of its filial generation, from transgenic animal head by the genetically modified part fragment of high-fidelity pcr amplification.This sheet segment length 1853bp comprises 4 exons of c-Ha-ras gene, also is to have comprised the position of easily undergoing mutation, i.e. 12,13 and 61 amino acids coding.Pcr amplification gained fragment is checked order behind the T-A clone, and carry out sequence alignment by DNAssist software.Sequence0 is an original series among the figure, as benchmark, and among Fig. 6-A, 96-98 base, 99-101 base 12,13 amino acid of encoding respectively; Among Fig. 6-B, 510-512 alkali yl coding the 61st amino acids.As seen, all do not undergo mutation in these 3 responsive sites.
Embodiment
Embodiment 1: the separation of anthropogenic proto-oncogene c-Ha-ras DNA
1. the structure of anthropogenic proto-oncogene c-Ha-ras DNA
The present invention relates to integrate in the genome making method of the rodents transgenic animal that contain anthropogenic proto-oncogene c-Ha-ras DNA.The used transgenosis of the present invention (Transgene) is the c-Ha-ras gene, and total length 6.5kb contains 4 exons, contains promotor, regulating and controlling sequence and the polyA signal sequence of this gene itself.It comes from the normal human blood tissue, and than the short 0.4kb (Fig. 1) of the used transgenosis of preceding bibliographical information, its full length sequence is seen Fig. 2.
2. the separation of anthropogenic proto-oncogene c-Ha-ras DNA
In bibliographical information before, transgenosis c-Ha-ras DNA obtains to adopt the mode that makes up the library to carry out (Sekiya T et al, 1984; 1985), the initiate dna of isolated genes is from a malignant melanoma Japanese patient.Because 12,13 and 61 amino acids of this proto-oncogene are easily undergone mutation, and often cause the activation of this gene after the sudden change, and further bring out canceration.And often undergone mutation from the DNA of tumor tissues, this is unfavorable to further microinjection.In the former bibliographical information, adopt molecular biology method transformation more, obtain the complete transgenosis of not undergoing mutation, increase more workload.
Because library construction is complexity, more laborious work.And round pcr is nowadays very ripe, so the present invention adopts PCR method segmentation amplification, T-A cloning and sequencing, is spliced into the strategy of complete genome then.According to sequence signature, three sections were separated (Fig. 3) before, during and after gene was divided into.Isolation of genomic DNA from normal human blood at first, adopting PCR method 5 ' the end length that increases respectively then is the fragment of 1991bp, the primer of amplification is to being Pri.F6506:gct
AagcttGgtcccggcggatcccagcctttc and Pri.R371:gagaattccagcctctccctggtacctctcatgcc; For the ease of connecting, in front end primer Pri.F6506, added restriction enzyme site HindIII; The fragment of the 1853bp in stage casing, the primer of amplification is to being Pri.F4886:gct
AagcttGgcaggtggggcaggagaccctgtag has added the HindIII site in primer, and Pri.RN1735:gc
GaattcCtgcggtcagcagcctcccttgtgcc has added the EcoRI site in primer; And the 3180bp fragment of 3 ' end, the primer that amplification is adopted is to being Pri.FN1735:ac
GaattcTcctgacgcaggtgagggggactc has added EcoRI in primer, and Pri.RXN (E): gct
GaattcAatgagggatccctggagggatgcctggtg has also added the EcoRI site in primer.The 1853bp fragment 5 ' end of amplification contains the KpnI site, and at 3 ' end, adds the EcoRI site.This amplified fragments is cut with KpnI+EcoRI is two, reclaimed the fragment of 1400bp, insert in same two pUC19 carrier of cutting, obtain first intermediate carrier pUC19-1400.With the 1991bp fragment HindIII+KpnI of amplification, insert and use intermediate carrier pUC19-1400 then, obtain the second intermediate carrier pUC19-3300.The fragment that obtains 3180bp with increasing is downcut once more with EcoRI behind the T-A cloning and sequencing, reclaims purifying, and with pUC19-3300 EcoRI linearizing, and, obtain to contain total length transgenosis c-Ha-ras carrier pUC19-6500 with the insertion of 3180 fragments.Because the segmental two ends of 3180bp are the EcoRI site, so need the direction of inserting is identified, the plasmid that direction of insertion is correct is preserved and is used for genetically modified amplification.
Embodiment 2: the importing of anthropogenic proto-oncogene c-Ha-ras DNA
Known to those skilled in the art, the process of producing transgenic animal by microinjection (Microinjection) comprises following a plurality of step: the female mouse of (1) sterile male mouse of preparation and false pregnancy; (2) super ovulation; (3) results zygote; (4) preparation of target DNA; (5) target DNA is imported zygote; (6) transplanting of zygote; (7) head builds the detection of target DNA integration situation among the mouse Founder; (8) by cross breeding transgenic mice strain.In the present invention, micro-injection method all carries out according to standard method, so do not do more narration.But the present invention has set up a kind of suitable method of dna solution being injected the microinjection pin in implementation process, and this method can be finished the filling of dna solution about 2 minutes, very fast.This technology has advanced the microinjection progress of work (biotechnology communication, 2007 04 phases) greatly.
Embodiment 3: the selection of transgenic animal
Any suitable rodent can be used as receptor to be used to produce " transgenic animal " of the present invention.Obviously, " transgenic animal " also the comprise transgenic animal newborn infant who is produced, young filial generation, fully-developed animal and embryo thereof; And the newborn infant of the filial generation of transgenic animal, young filial generation, fully-developed animal and embryo thereof.The example of transgenic animal and filial generation thereof comprises mouse, rat, cavy or the like, and preferred rodent is a mouse, as the C57 mouse.This is that primer C57 is sophisticated inbred lines, and genetic background is clear, and is stable, and its zygote being fit to do microinjection, be easy to the analogue formation animal relatively.
Embodiment 4: the phenotype of transgenic animal
The head that the present invention obtains builds mouse and has new phenotype.Japan rasH2 belongs to the induction type model, its outward appearance and body weight and normal mice almost are as good as (http://www.rash2.com/-phenotype), generally only when giving positive carcinogens, just induce the generation tumour, the probability of spontaneous knurl very low (＜1.1%), time of origin (8～27 weeks in evening, the skin squamous-cell papilloma, http://www.rash2.com/-background data).And the head that this research obtains built about mouse birth 2 weeks of back, i.e. the visible knob of spontaneous generation (Fig. 4) at the back, and fast growth, 6 all left and right sides the maximum reach 1 * 1cm, and develop into many places by a place.That is to say that with comparing of Japan, the cancer transgenic models that this research obtains has become spontaneous type by induction type, the time of generation is than early.After 1 year, tangible knurl (Fig. 5) promptly occur at neck in this model birth, it is extremely rapid to grow.After the dissection, the visible many places of lung's naked eyes tumour (Fig. 5-B, D).
In the research of the transgenic animal model of relevant ras gene, the model that two weeks after birth, promptly occur knob do not appear in the newspapers as yet (Xi Tao etc., 2001).Through continuing further investigation, be expected to realize: (1) obtains an animal model that can be used for the drug induced carcinogenesis safety evaluation.This is the original intention of carrying out this research, promptly substitutes traditional big mouse, be used to estimate new drug carinogenicity, screening cancer therapy drug, detect the carinogenicity of the property inserted medicine equipment etc.Substitute traditional big mouse carinogenicity experiment can shorten the time greatly (6 months vs2), saving funds with transgenic animal model, reduce the consumption of animal, meet in the world " optimize, substitute, reduce " 3R principles and requirements about animal welfare.Therefore, this animal model has important application value.(2) this model has new characteristic, can be used as a new animal model for cancer, is used for the research of cancer genesis mechanism.Mainly show: this model is a spontaneous type, and time of origin is more Zao than Japanese rasH2 model, and this has hinted that this model may be more more responsive than rasH2 for carcinogenic reaction, is more suitable for being used for the research of cancer genesis mechanism aspect.The underlying causes that takes place in view of human cancer is " human body itself and the long-term results of interaction of environmental factors ", and generally speaking, it is very low that human body contacts the strong carcinogenic probability of similar heavy dose, so have reason to believe, it is truer near human body that the data ratio of the relevant cancer mechanism that the spontaneous type model draws brings out type.(3) appearance of early stage knob means that more more responsive than former same class model, this is of paramount importance when being used for the carinogenicity experiment.
Embodiment 5: the mating of transgenic animal with building is
Adopt following scheme mating to build to be: for female head build mouse then with normal male mouse mating, select age in week in 10 weeks, have mating experience, healthy and strong C57 mouse mating with it.Live together, begin to check pregnant situation after 1 week, if find that pregnancy is about to female mouse and raises separately, until production.If build mouse, then select more than 8 weeks healthy and strong female mouse mating with it for male head.Each only first offspring's separate marking of building mouse is treated as a system.Detect to find transgenosis can be in the offspring genetic stability, litter size amount and normal mouse are as good as, and generally can successfully feed, the rare maternal instinct bad phenomenon such as son of eating.It is to lay the foundation that these advantageous feature are built for next step large-scale breeding.
Embodiment 6: genetically modified sudden change detects
Build mouse, comprise the tail point tissue extraction DNA of its filial generation, from transgenic animal head by the genetically modified part fragment of high-fidelity pcr amplification.This sheet segment length 1853bp comprises 4 exons of c-Ha-ras gene, also is to have comprised the position of easily undergoing mutation, i.e. 12,13 and 61 amino acids.The primer of amplification is to being Pri.F4886:gct
AagcttGgcaggtggggcaggagaccctgtag and Pri.RN1735:gcgaattcctgcggtcagcagcctcccttgtgcc (EcoRI).Pcr amplification gained fragment is checked order behind the T-A clone, and carry out sequence alignment by DNAssist software.The result shows these several sites all do not undergo mutation (Fig. 6).
Embodiment 7: the life cycle of transgenic animal
The life cycle of cancer transgenic animal model is shorter, the probability rising of spontaneous tumor generally occurs in time more than 6 months, and is easily dead.Among the present invention, the life cycle that head builds mouse reached about 1 year, and general state is normal.
Sequence table
＜110〉Nat'l Pharmaceutical ﹠ Biological Products Control Institute
＜120〉contain the making method and uses thereof of the transgenic mice of anthropogenic proto-oncogene c-Ha-ras
<160>1
<170>PatentIn?version3.4
<210>1
<211>6506
<212>DNA
＜213〉people
<400>1
Claims (10)
1. people's proto-oncogene c-Ha-ras, its sequence is shown in SEQ ID NO:1.
2. people's proto-oncogene c-Ha-ras as claimed in claim 1, it contains 4 codings exon, promoter region and polyadenylic acid tailing signal sequence polyA.
3. people's proto-oncogene c-Ha-ras as claimed in claim 2, wherein said promotor is the promotor of rodents villin promotor or cytomegalovirus CMV; Describedly add the polyA signal sequence that tailer sequence is SV40 or BGH.
4. the host cell that contains people's proto-oncogene c-Ha-ras as claimed in claim 1.
5. the tissue that forms by host cell as claimed in claim 4, organ.
As claim 4 or 5 described host cells, tissue or organ in the carcinogens screening or estimate, press down purposes in the safety evaluation of screening, medicine of cancer thing.
7. as claim 4 or 5 described host cells, tissue or the purposes of organ in toxicology.
8. as claim 4 or 5 described host cells, tissue or the purposes of organ in immunotoxicology.
9. contain the method for the rodents zygote of people's proto-oncogene c-Ha-ras as claimed in claim 1 in the preparation genome, step is:
(1) separates acquisition people proto-oncogene c-Ha-ras gene;
(2) obtain rodents zygote by super ovulation, mating, zygote screening and separating step;
(3) by micro-injection method the c-Ha-ras gene is injected zygote, obtain to contain the rodents zygote of people's proto-oncogene c-Ha-ras.
10. contain the method for the rodents transgenic animal of people's proto-oncogene c-Ha-ras as claimed in claim 1 in the preparation genome, step is:
(1) makes the male mouse of vasoligation, allow itself and normal heat mouse mating then, obtain the false pregnancy mouse;
(2) with zygote directly transplanting as claimed in claim 9 or at CO
2Be implanted in the uterine tube of false pregnancy mouse during incubated overnight to 2 cell in the incubator;
(3) allow false pregnancy mouse cub, treat behind breast to detect by molecular biology method and obtain the transgenic positive filial generation, promptly head builds mouse;
(4) after head builds the mouse sexual maturity, carry out mating and build and be, obtain this transgenic animal system.
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Cited By (3)
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| CN112005960A (en) * | 2019-05-31 | 2020-12-01 | 武汉科技大学 | Method for automatically breeding pregnant female mice |
| CN114134178A (en) * | 2021-12-10 | 2022-03-04 | 中国食品药品检定研究院 | Carcinogenic mouse model and establishing method and application thereof |
| CN117778467A (en) * | 2023-12-26 | 2024-03-29 | 上海药康生物科技有限公司 | Construction method and application of HRAS humanized mice for carcinogenicity evaluation |
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| BIRREN B ET AL: "序列号：AC137894", 《GENBANK》 * |
| HIROYUKI TSUDA ET AL: "High susceptibility of human c-Ha-ras proto-oncogene transgenic rats to carcinogenesis: A cancer-prone animal model", 《CANCER SCI》 * |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112005960A (en) * | 2019-05-31 | 2020-12-01 | 武汉科技大学 | Method for automatically breeding pregnant female mice |
| CN114134178A (en) * | 2021-12-10 | 2022-03-04 | 中国食品药品检定研究院 | Carcinogenic mouse model and establishing method and application thereof |
| CN117778467A (en) * | 2023-12-26 | 2024-03-29 | 上海药康生物科技有限公司 | Construction method and application of HRAS humanized mice for carcinogenicity evaluation |
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