CN103952441B - The construction method of 1.3 copy C genotype HBV transgenic mices - Google Patents
The construction method of 1.3 copy C genotype HBV transgenic mices Download PDFInfo
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Abstract
The invention provides the construction method of a kind of 1.3 copy C genotype HBV transgenic mices.Described method is to prepare site-directed integration 1.3 by blastaea injection ES cell to copy C genotype hepatitis B virus (Hepatitis B virus, HBV) transgenic mice.First build site-directed integration 1.3 and copy the expression vector of c-type HBV, then use electricity transduction method for transformation in recombinant plasmid transformed to ES cell, the ES cell of site-directed integration restructuring is obtained after screening and cultivation, again the ES cell of site-directed integration restructuring is expelled in blastaea after being digested to individual cells, then blastaea is transplanted in pseudo-fetus dams uterus.The method at home and abroad takes the lead in establishing the transgenic mice of 1.3 copy c-types HBV, is applied to targetedly in hepatitis B Related Research Domain, for China's hepatitis B prevention and Therapy study more preferably animal model.
Description
Technical field
The present invention relates to animal model and build field.Construction method more particularly, to a kind of 1.3 copy C genotype HBV transgenic mices.
Background technology
It is the interior significant problem affecting human health of global range that hepatitis B virus (hepatitis B virus, HBV) infects.The chronic progressive external inflammation pathological changes caused by HBV infection, usually develops into liver cirrhosis, hepatocarcinoma and ultimately results in death.According to World Health Organization, the whole world about 2,000,000,000 people once infected HBV, and wherein 3.5 hundred million people are Patients with Chronic HBV Infection, and the most dead number is about 1,000,000～2,000,000.China is always the district occurred frequently of HBV infection, and up-to-date data shows: the existing Patients with Chronic HBV Infection of China is about 93,000,000 examples, and wherein chronic hepatitis B patient about 20,000,000 example, accounts for the last 1/5 of Patients with Chronic HBV Infection.Patients with Chronic HBV Infection and chronic hepatitis B patient, owing to carrying a large amount of HBV, are the main sources of infection of HBV.China the most all puts into substantial amounts of funds and manpower physical resources and for the treatment of such crowd and stops HBV being propagated further in crowd, brings the most serious economy and burden on society to China, makes this most extremely limited medical resource more nervous.Go deep into and multi-level HBV infection especially chronic HBV infection and the correlational study of chronic hepatitis B to this end, carry out, affect, for capturing this, the disease that nearly 100,000,000 populations of China are healthy, safeguard the healthy significant of China's population.
In the correlational study of hepatitis B, hepatitis B animal model has very important status, and hepatitis B animal model is applied to the at all levels and various aspects of the therapeutic evaluation etc. of the immune state change of body machine-processed from HBV infection, infected, HBV Immune escaping mechanism, the gene therapy of HBV and HBV medicine in infected body.Thus setting up and the key selecting often to become HBV research work of suitable animal model, research work is played a multiplier role.To this end, many scholars are the most once devoted to set up various types of hepatitis B animal model.Belonging to Hepadnaviridae yet with mankind HBV, have stronger species specificity, lower of natural conditions infect people and non-human primates (such as chimpanzee etc.), it is established that can wide variety of animal model not a duck soup.Chimp model is applied because its volume is relatively big, cost is high and is limited it by ethics constraint etc.；Tree-model is then low due to its efficiency of infection, is only capable of causing of short duration low-grade infection, and virus titer is the lowest, and is difficult to popularization and application；The animal model of duck hepatitis-B, then there is its pharmacokinetics and defect, marmot that immunology genetic background is very different with the mankind only originate in North America, transport difficult and expensive, thus limit the extensive application of these animal models, and then limit the correlational study to pathogenesis of hepatitis B, immunopathogenesis, medicine and immunization therapy.
In recent years, owing to heredity and immunity background understand, are prone to the advantages such as raising, mice is gradually paid close attention to by research worker, becomes the optimum selection building HBV animal model.Research worker establishes people-Hepar Mus chimera HBV mouse model, gene HBV transfected mouse model and HBV transgene mouse model.
But, although people-Hepar Mus chimera HBV mouse model can be described as the most best HBV infection duplicating model, but owing to this model does not has immune function, thus immune-mediated virus sweep and the research of hepatitis immunity mechanism of causing a disease can not be carried out.Gene HBV transfected mouse model is owing to being transient hbv replication model, thus is only used for the research of acute HBV infection, is not suitable for chronic HBV infection and chronic hepatitis B research.Additionally, due to injection dosage is too big (being equivalent to the total amount of mouse systemic body fluid), the damage causing mouse liver and other organs can not be ignored.Current existing HBV transgenic mice there is also significantly defect.First, current existing HBV transgenic mice, owing to setting up not by by the approach of HBV infection, therefore cannot be used for carrying out HBV and enter infected body and the HBV distribution research in body；Secondly, current existing HBV transgenic mice is in immune tolerance state to HBV virus antigen, and body can not produce the immunoreation to HBV, does not the most produce hepatitis B pathological changes.Above reason result in the disappearance of the animal model that can be used for chronic HBV infection and the nonimmune tolerance of chronic hepatitis B, objectively have impact on carrying out in a deep going way of correlational study.Hence set up controlled, support HBV In vivo infection and can cause being similar to clinical hepatitis B pathological changes, the HBV mouse model of nonimmune tolerance becomes understands hepatitis B pathogenesis in depth, the development of hepatic lesions and lapse to mechanism, the research and development of anti-hbv drug and evaluate the important key factor of new hepatitis B immune therapy.
Additionally, owing to the HBV antiviral therapy that current clinical treatment hepatitis B is widely used exists a lot of not enough, as interferon can cause patient's leukocyte and platelet to decline, may increase the weight of hepatic injury, likely teratogenesis or hinder childhood development, the not untoward reaction being resistant to medicine etc.；Nucleoside analog then produces drug resistance owing to causing the HBV gene in host to be undergone mutation.It is not satisfactory that these factors cause HBV infection clinically to treat the therapeutic effect of especially chronic HBV infection and chronic hepatitis B, expect that new Therapeutic Method comes out clinically, to reach to control or reverse process and the progress that hepatitis B especially chronic HBV infection and chronic hepatitis B cause a disease, until it reaches thoroughly cure the purpose of hepatitis B.Immune modulating treatment is expected to become the important means for the treatment of chronic hepatitis B.Research shows, the T cell in HBV chronic infection's body is triggered by HBV and commits suiside, and this may is that the key determining that HBV cannot remove in HBV chronic infection's body.
It addition, hepatitis B virus (Hepatitis B virus, HBV) is a kind of hepadnavirus, the diseases such as various acute, chronic hepatitis, liver cirrhosis can be caused, and have very close relationship with hepatocarcinoma.According to the difference of HBV gene sequence, HBV strain can be divided into different genotype, have determined that A～H8 genotype at present.There is the most close relation in the aspects such as HBV gene type and its epidemiological features, pathogenic, disease treatment and prognosis.Different genotype is certain geographic regional property distribution, and the Major Epidemic strain of China has Type B and c-type.And genotype C the most all has popular, especially with northern area Flow Behavior master, some cities are even as high as 69%.
Owing to HBV has the host specificity of height, only infecting people and minority primate, the not common laboratory animal such as infecting mouse, the research about HBV and hepatitis B is very limited.The foundation of transgenic mice, brings hope for the basic research of hepatitis B and the exploitation of anti-hepatic-B virus medicine.But HBV full-length genome list copy or two copy transgenic animal experiments show that the duplication of HBV and expression efficiency are the lowest, have had a strong impact on it and have extensively applied.Studies in China mostly is D type transgenic mice at present.And it is less for state's epidemic strain c-type transgenic mice research.The most extremely it is necessary to set up a kind of c-type HBV gene efficient replication and the transgenic mice of expression, for China's hepatitis B prevention and Therapy study more preferably animal model.But, the c-type transgenic mice that this laboratory is prepared by the method for micro-procaryotic injection, in ImmunohistochemistryResults Results, show that F0 generation all has HBsAg to express to F3 for hepatic tissue and the nephridial tissue of positive mice, and be plasmotype, show that this gene can be expressed and can be passed on.But mice serum and urine HBsAg and HBeAg are difficult to detect, serum quantitative fluorescent PCR displays that HBV DNA is low duplication expression, and copy number is 102～103Copy/mL.
Summary of the invention
The technical problem to be solved is the technical deficiency overcoming existing hepatitis B research transgenic animal model to exist, a kind of c-type hepatitis B diseases research animal model that can stably pass on and express, i.e. efficient replication are provided and express the construction method of c-type HBV transgenic mice.
It is an object of the present invention to provide the preparation method of a kind of 1.3 copy C genotype HBV transgenic mices.
The present invention also provides for the application of above-mentioned 1.3 copy C genotype HBV transgenic mices.
Above-mentioned purpose of the present invention is achieved through the following technical solutions:
The invention provides the construction method of a kind of 1.3 copy C genotype HBV transgenic mices, step is as follows:
S1. the foundation of embryonic stem cell line；
S2. 1.3 extraordinarily long segment c-type HBV plasmid electricity are transduceed to embryonic stem cell, through cultivating and puromycin screening obtains the embryonic stem cell of the positive, be enlarged cultivating；
S3. in positive embryos stem cell injection to blastula stage mouse eggs cell, the blastaea injected is transplanted to the uterus of pseudo-fetus dams, cultivates and obtain 1.3 copy C genotype HBV transgenic mices.
Wherein, the method for building up of embryonic stem cell line described in step S1 is as follows:
S11. taking the female pregnant Mus of C57BL/6J of conceived 3.5 days, disconnected neck is put to death；
S12. the pregnant Mus sterilization with ethanol, S11 put to death；
S13. open pregnant Mus abdominal cavity, repair removing fat and mesentery, cut bilateral uterine from fallopian tube root and lower end, uterus crotch, uterus bicornis is cut off at vagina；
S14. syringe sucks M2(purchased from Sigma Co., USA) as embryo washing water, the syringe needle of syringe is cut needle point before the use and is polished sterilizing；By syringe in one end intron uterine cavity in uterus, clamping, it is blown into embryo washing water, then the other end from uterus inserts syringe, is blown into embryo washing water；
S15. being taken out by above-mentioned embryo washing water and be placed in plate, plate is placed under anatomical lens, holds pipe in the month with mouth and draws a small amount of embryo washing water, by blastaea sucking-off；
The most then blastaea is put into KSOMaa embryo culture medium (purchased from U.S. Zenith
Biotech company) in, observe, select reasonable blastaea；
S17. the blastaea of selection is blown into the cell plates being vaccinated with feeder layer cells (offer of Beijing Si Saiyin company) in advance, uses Establishing Mouse Embryonic Stem Cell Line culture medium (purchased from Beijing Si Saiyin company) instead, every hole is put 1 blastaea；
S18. putting into incubator to cultivate, blastaea is gradually grown up, the most adherent, grows germinal layer and launches, and the inner cell of blastaea is constantly bred；
S19. cultivating after 4～6 days, the inner cell of blastaea becomes a reunion cylindricality or protruding cell mass, now can the inner cell mass of discrete blastaea, obtain embryonic stem cell line.
Described in step S2,1.3 extraordinarily long segment c-type HBV plasmids are gifted by No.302 Hospital, P.L.A., and sequence is as shown in SEQ ID NO.1.
The construction method of described 1.3 extraordinarily long segment c-type HBV plasmids is: pass throughSpe IWithApa IThe DNA fragmentation of 1.3 extraordinarily long segment c-types HBV is connected in pcDNA3.1 plasmid by two restriction enzyme sites, obtain 1.3 extraordinarily long segment c-type HBV plasmid schematic diagram as shown in Figure 1.
Described in step S2, the method for electricity transduction is as follows:
S21. it is individual cells by the embryonic stem cell line trypsinization of cultivation, add and terminate culture medium (Guangzhou academy of science provides with health research institute) termination digestion, centrifugal, remove supernatant, then with subtracting blood serum medium opti-MEM(purchased from Gbico company of the U.S.) suspend；
S22., suspension adds the c-type HBV plasmid needed for electricity transduction, mixing, transfers in electricity revolving cup, carry out electricity transduction with electroporation, be rapidly added cell culture medium (Guangzhou academy of science provides with health research institute) after electricity transduction, be then taped against in cell plates；
S23. second day, replace medium to the culture medium of puromycin-resistant, carry out every day changing liquid；
S24., after cultivating 5～7 days, the cell only having proceeded to plasmid just can grow into colony clone, and what picking screened respectively is cloned in cell plates, 1, each hole cell clone；
S25. second day, by passage to new cell plates, after again covering with, cell was used for extracting DNA and carries out PCR qualification, obtains positive colony；
S26. it is enlarged the cell of the positive colony obtained cultivating, obtains positive embryos stem line.
Described in step S3, the method for injection is as follows:
S31. the positive embryos stem line that electricity transduction obtains is digested resuspended one-tenth individual cells；
S32. punch on the zona pellucida of blastaea with laser rupture of membranes instrument, with entry needle, 10～20 pieces of positive embryos stem cell are got to inside segmentation cavity, then by embryo culture medium KSOMaa(purchased from Zenith Biotech company of the U.S.) cultivate 2～3 hours after, basis of microscopic observation blastaea form is recovered.
Pseudo-fetus dams described in step S3 obtains by the following method: KM dams and KM are ligatured public Mus and carries out mating raising according to female-male proportion 2:1, select cloudy bolt and vaginal orifice swelling, ruddy dams secondary morning, it is pseudo-fetus dams, it is denoted as 0.5 day pseudo-fetus dams, and blastaea injection transplantation was in 2.5 days pseudo-fetus dams.
Present invention also offers above-mentioned 1.3 copy C genotype HBV transgenic mice application in terms of hepatitis B Related Research Domain.
More specifically, described application refers to the application in research body immune system anti-hepatitis virus infection, immunity pathogenic course and in terms of anti-hepatic-B virus medicine exploitation of the 1.3 copy C genotype HBV transgenic mices.
Used by the present invention, C57BL/6J Mus is preserved by this laboratory.The key property of C57BL/6J Mus is: (selecting from " experimental zoology ", chief editor: Hao Guangrong, publishing house of The 2nd Army Medical College, see this book page 105)
(1) immunological characteristic: IgG was slowly increased before 20 monthly ages, and IgG2b is high level, and IgG is low value.Aseptic raising more common raiser IgG absolute magnitude is low.IgG is high level, can be more than 800 μ g/mL after 12 monthly ages of some individualities.The IgM of aseptic raising is higher.Cellular immunity, may be less with spontaneous tumor relevant with increasing less reduction in age.It is easier to induce immunologic tolerance.Interferon yield is higher.The factor (Pertussis susceptible to pertussis
HSF) sensitive；
(2) morphological characteristic: in new cub, female 16.8%, male 3% is ommatidium or anophthalmia disease；There is hind leg many toes disease in the 0.6% of new cub；
(3) physiology characteristic: high addicted to Alcoholic, adrenal gland's stocking up lipids is few；Hero Corium Mus skin is migrated to the female Mus of homology, after 20 days, occurs that row tears open；This is because there is male antigen during histocompatibility is former, C57BL/6J mice is that mice is the most notable compared with other；Amyloidosis disease is easily caused after injection casein；Can induce with cortisone and occur that the probability of cleft palate is 20%, sensitive to tubercule bacillus, mouse pox virus is had certain drag；
(4) carcinogenesis rate: breast carcinoma sends out (breast carcinoma rate is 0～1%) less；It is difficult to carcinogenic with carcinogen.The aged spontaneous rate of Mus lymphoma is 20～25%；Female murine leukemia is 7～16%；After roentgen radiation x, hepatocarcinoma incidence rate is high；
(5) life-span: for up to 1200 days；Average female Mus 692 days, male Mus 676 days.
It is black that the present invention selects C57BL/6J Mus to be primarily due to its hair color, and during blastaea injection, embryonic origin is the KM Mus of white hair, thus without being detected the chimeric rate of Chi-meric mice by other detection method, directly by hair color it may determine that the rate of being fitted together to.
Less currently for the research of domestic epidemic strain c-type HBV transgenic mice.The present invention utilizes blastaea injection to take the lead in setting up one at home and can stably pass on and efficient replication expression c-type HBV transgenic mice, provides more preferably animal model for China's hepatitis B prevention and Therapy study.The present invention builds transgenic mice by blastaea injection ES cell method, and c-type HBV gene can pinpoint restructuring ES cell, and (structural representation of fixed point restructuring is as shown in Figure 2.Gene targeting technology is also called gene targeting, refer to build the integration vector of the DNA fragmentation containing homologous sequence, by various method for transformation, make to occur between exogenous DNA array and target sequence homologous recombination, thus target DNA sequence fixed point is destroyed, by a series of screening means, finally give the cell that orientation converts.One of them method of Gene targeting technology is just introduced into recombinase Flp+Cre system, homologous recombination is carried out by replacement vector in embryonic stem cell (ES cell), introduce selectable marker gene to genome target site, and introduce FRT and the Loxp site being collectively aligned in its both sides respectively.Article two, transcribing of target gene can not be disturbed in homologous chromosome all FRT and Loxp sites with FRT and Loxp site and introducing.
Being shown by substantial amounts of experiment, the efficiency of the positive C genotype HBV transgenic mice obtained according to construction method of the present invention is the highest, and can stably pass on.Through experimental verification repeatedly, the mouse chimera rate that the present invention is obtained by blastaea injection ES cell has 40%～60%, substantially increases the transgene efficiency of mice.It addition, the method that the present invention uses blastaea injection ES cell, HBV-C gene can pinpoint restructuring ES cell, and the c-type HBV allophenic mice positive rate obtained is higher, and positive rate is 50%, hence it is evident that the c-type HBV transgenic mice obtained higher than prior art.
Additionally, transgenic mice of the present invention has the feature that the 1.3 C genotype HBV transgenic mices copied are different that turns from tradition, duplication and the expression efficiency of the HBV of this model are higher, for research hepatitis B pathogenesis further, the development of hepatic lesions and lapse to Mechanism Study, provides preferable animal model for pathological change and the body immune system mechanism of action in viral infection resisting and immunity pathogenic course of body after c-type HBV infection.Setting up of this model will fill up the blank lacking this type of animal model both at home and abroad, make the research means being correlated with abundanter, also the research and development for anti-hbv drug provide more advanced appraisement system and technology platform, for setting up the immunotherapy method of hepatitis B make an appraisal a kind of brand-new thinking of offer and research platform, this transgene mouse model can be utilized to carry out research and the exploratory study of hepatitis B immune treatment new method of hepatitis B immune mechanism of causing a disease, and establish solid foundation for the follow-up industrialization realizing this new hepatitis B diseases model and batch supply, promote China's treating hepatitis B, the raising of research level.
The method have the advantages that
Instant invention overcomes the tradition of hepatitis B research and turn the C genotype HBV transgenic mice hbv replication of 1.3 copies and the technical problem such as expression efficiency is relatively low, provide a kind of method utilizing blastaea injection to build transgenic mice, the method is capable of c-type HBV site-directed integration transgenic mice, acquisition can stably be passed on and efficient c-type hepatitis B diseases model, i.e. efficient replication and the construction method of expression c-type HBV transgenic mice, filled up the blank of domestic and international this type of animal model of shortage.
Additionally, it is C57BL/6 mice that the present invention builds the ES cell derived of transgenic mice use, hair color is black, and blastaea source is KM mice, hair color is white, and after ES cell infusion to blastaea, the hair color of the mice born is to be partly ash or black, and the next generation can be genetic to, thus can identify chimeric rate simply by hair color.And the strong KM Mus of selected reproduction ability is cooked pseudo-fetus Mus, farrowing rate is high, and maternal instinct is good.
Construction method of the present invention builds the transgenic mice obtained can reflect clinical hepatitis B generation, evolution comprehensively, truly, for hepatitis B pathogenesis, the development of hepatic lesions and lapse to Mechanism Study, body immune system viral infection resisting and immunity pathogenic course in study on mechanism and the research and development of anti-hbv drug and evaluate provide ideal animals model, for setting up the immunotherapy method of hepatitis B and making an appraisal and provide a kind of brand-new thinking and research platform, promote the raising of China's treating hepatitis B research level.
Accompanying drawing explanation
Fig. 1 is 1.3 extraordinarily long segment c-type HBV plasmid schematic diagrams.
Fig. 2 is the structural representation of c-type HBV gene fixed point restructuring ES cell.
Fig. 3 is that construction method of the present invention builds the positive Chi-meric mice obtained.
Detailed description of the invention
Further illustrate the present invention below in conjunction with the drawings and specific embodiments, but the present invention is not limited in any form by embodiment.Unless stated otherwise, the present invention uses reagent, equipment and method are the art conventional reagent, equipment and method.
Unless stated otherwise, C57BL/6J mice used by the embodiment of the present invention and KM mice are commercial.
Experimental example
1
Build
1.3
Copy
C
Genotype
HBV
Transgenic mice
1, the foundation that embryonic stem cell (ES cell) is
(1) taking the female pregnant Mus of C57BL/6J of conceived 3.5 days, disconnected neck is put to death.
(2) the pregnant Mus sterilization put to death by S11 with 75% ethanol, squirts whole body as far as possible.
(3) open pregnant Mus abdominal cavity, repair removing fat and mesentery, cut bilateral uterine from fallopian tube root and lower end, uterus crotch, uterus bicornis is cut off at vagina.
(4) syringe sucks M2(purchased from Sigma Co., USA) as embryo washing water, the syringe needle of syringe is cut needle point before the use and is polished sterilizing；By syringe in one end intron uterine cavity in uterus 2～3mm, clamping, it is blown into embryo washing water, then the other end from uterus inserts syringe, is blown into embryo washing water.
(5) taking-up of above-mentioned embryo washing water being placed in plate, plate is placed under anatomical lens, holds pipe in the month with mouth and draws a small amount of embryo washing water, by blastaea sucking-off.
(6) then blastaea is put in KSOMaaa embryo culture medium (purchased from Zenith Biotech company of the U.S.), observe, select reasonable blastaea.
(7) blastaea of selection is blown into 4 orifice plates or 24 orifice plates of feeder layer cells (offer of Beijing Si Saiyin company) are provided in advance, use Establishing Mouse Embryonic Stem Cell Line culture medium (purchased from Beijing Si Saiyin company) instead, every hole is put 1 blastaea.
(8) putting into incubator to cultivate, blastaea is gradually grown up, the most adherent, grows germinal layer and launches, and the inner cell of blastaea is constantly bred.
(9) cultivating after 4～6 days, the inner cell of blastaea becomes a reunion cylindricality or protruding cell mass, now can the inner cell mass of discrete blastaea, obtain embryonic stem cell line.
2, the extraordinarily long segment c-type HBV plasmid of 1.3 used by the present embodiment is gifted by No.302 Hospital, P.L.A., and sequence is as shown in SEQ ID NO.1.
The construction method of described 1.3 extraordinarily long segment c-type HBV plasmids is: pass throughSpe IWithApa IThe DNA fragmentation of 1.3 extraordinarily long segment c-types HBV is connected in pcDNA3.1 plasmid by two restriction enzyme sites, obtain 1.3 extraordinarily long segment c-type HBV plasmid schematic diagram as shown in Figure 1.
3,1.3 copy C genotype HBV electricity are transduceed embryonic stem cell (ES cell)
(1) it is individual cells by the embryonic stem cell line (12 orifice plate one holes cover with) cultivated with trypsinization, add and terminate culture medium (Guangzhou academy of science provides with health research institute) termination digestion, transfer to 1000rpm in 15mL centrifuge tube and be centrifuged 5 minutes, remove supernatant (removing clean the most completely), then with the opti-MEM(of 100 L purchased from Gbico company of the U.S.) suspend, transfer in centrifuge tube (EP) pipe of 1.5mL；
(2) 1.3 extraordinarily long segment c-type HBV plasmid 5 g that electricity turns required are added, mix gently with liquid-transfering gun, it is then transferred in electricity revolving cup, carry out electricity with electroporation to turn, electricity is rapidly added cell culture medium (Guangzhou academy of science provides) with health research institute after turning, be then taped against in cell plates (6 orifice plate 1 hole).
(3) second days, replace medium to the culture medium (puro using 1 g/mL screens) of resistance, carry out every day changing liquid (resistance).
After (4) 5～7 days, the cell only having proceeded to plasmid just can grow into colony clone, and what picking screened respectively is cloned in 96 orifice plates, 1, each hole cell clone.
(5) second days, by passage to 48 orifice plates, after again covering with, most cells may be used for extracting DNA and carries out PCR qualification, remaining cell continue to cultivate until qualification result out.
(6) it is enlarged the cell of the positive colony obtained cultivating, obtains positive embryos stem line.
After electricity is transduceed, 1.3 copy c-type HBV gene can pinpoint restructuring ES cell, and the schematic diagram of fixed point restructuring ES cell is as shown in Figure 2.
4, positive embryos stem cell blastaea injection
Within (1) 3.5 day, KM dams obtains blastaea by rushing bilateral uterine.
(2) the positive embryos stem line that electricity transduction obtains is digested resuspended one-tenth individual cells.
(3) punch on the zona pellucida of blastaea with laser rupture of membranes instrument, with entry needle, the positive embryos stem cell of 15～20 pieces is got to inside segmentation cavity, then, after cultivating 2～3 hours by KSOMaa embryo culture medium (purchased from Zenith Biotech company of the U.S.), basis of microscopic observation blastaea form is recovered.
5, blastaea is transplanted
(1) preparation of pseudo-fetus dams
KM Mus fertility is strong, and farrowing rate is high, and maternal instinct is good, therefore selects KM Mus to prepare pseudo-fetus Mus.
The preparation method of pseudo-fetus dams is as follows: KM dams and KM are ligatured public Mus and carries out mating raising according to female-male proportion 2:1, select cloudy bolt and vaginal orifice swelling, ruddy dams secondary morning, it is pseudo-fetus dams, is denoted as 0.5 day pseudo-fetus dams, and blastaea injection transplantation was in 2.5 days pseudo-fetus dams.
The blastaea injected is transplanted to the uterus of the pseudo-fetus dams of 2.5 days, cultivates and obtain 1.3 copy C genotype HBV transgenic mices.
6, result statistics
Result injects blastaea 221 pieces altogether, survives 180 pieces, is injected into motility rate 81.45%.The female Mus of co-transplantation pseudo-fetus 14,10 pregnancies, pseudo-fetus pregnancy rate 71.4%；Farrowing 10, wherein eaten 2 second day puerperal, 8 F0 of survival are for mice.
Experimental example
2
Embodiment
1
Preparation
1.3
Copy
C
Type
HBV
The qualification of transgenic mice
1, the statistics of mouse chimera rate
(1) judge the chimeric rate of mice according to mice hair color: owing to ES cell derived is in the C57BL/6J Mus of black hair, and blastaea derives from the KM Mus of white hair, thus judge the chimeric rate of mice according to the percentage rate of birth mice black wool.
(2) Chi-meric mice (F0 generation) obtained and normal KM Mus are mated.If the F1 generation mice obtained is Chi-meric mice, it was demonstrated that this F0 has reproduction to be fitted together to for Chi-meric mice, can be genetic to the next generation.
2, the hepatitis B surface antigen (HBsAg) during ELISA detects serum of transgenic mice and urine and hepatitis B virus e antigen (HBeAg), detection method is as follows:
(1) detection (ELISA method) of hepatitis B surface antigen (HBsAg)
Utilize hepatitis B virus surface antigen diagnostic kit (purchased from Shanghai Kehua Bio-technology Co., Ltd) production licence number: Shanghai 20110148, authentication code: traditional Chinese medicines quasi-word S10910113, product batch number: 20130105 detect, detection method is carried out with reference to description.
Detection concrete operations are as follows:
S1. configuration working concentration cleaning mixture, carries out 25 times of dilutions with pure water；
S2. according to requirement of experiment (9 holes, a blank control wells, two negative control holes, two Positive control wells, 4 Chi-meric mice samples), the reaction lath in 9 holes is selected；
S3. 4 Chi-meric mice samples and negative control, the positive control (what positive control of negative control is that test kit carries) (reserved positive control 2 hole, negative control 2 hole, blank 1 hole) in reacting hole of 75 L it are separately added into.Then cover Sptting plate with mounting paper, Sptting plate is placed in 37 DEG C and hatches 60min；
S4. take out Sptting plate, tear mounting off, in the hole adding sample to be tested and positive and negative comparison, add 50 L enzyme conjugates (test kit carries), blank control wells is not added with, shake 10s gently, cover reaction bar with mounting paper, Sptting plate is placed in 37 DEG C and hatches 30min；
S5. wash plate: take out Sptting plate, tear mounting off, discard liquid in hole, fill each hole with the working concentration cleaning mixture of step S1 configuration, stand 30～60s, dry, after being repeated 5 times, clean absorbent paper pats dry；
S6. at porose interior addition developer A, each 50 L of developer B, mixing；Shake 10s gently, cover reaction bar with mounting paper, Sptting plate is placed in 37 DEG C and hatches 30min；
S7. at porose interior addition 50 L stop buffer, oscillating reactions plate 5s；Microplate reader reading, wavelength is 450nm and 630nm dual wavelength, first returns to zero by blank well, then reads each hole OD value.
Result judges: COV=negative control meansigma methods+0.1.As sample to be tested OD value >=COV, it is judged that for the positive；When sample to be tested OD value is < during COV, it is judged that for feminine gender.
(2) hepatitis B virus e antigen (HBeAg) (ELISA method)
Hepatitis B virus e antigen diagnostic kit (purchased from Shanghai Kehua Bio-technology Co., Ltd) production licence number: Shanghai food medicine prison tool produces is permitted No. 20030916, equipment registration number: state's food medicine prison tool (accurate) word 2012 the 3400740th, product standard number: YZB/ state 2142-2012 product batch number: 20121206 detect, detection method is carried out with reference to description.
Detection concrete operations are as follows:
S1. configuration working concentration cleaning mixture, carries out 25 times of dilutions with pure water；
S2. according to requirement of experiment, the reaction lath (9 holes, a blank control wells, two negative control holes, two Positive control wells, 4 Chi-meric mice samples) in 9 holes is selected；
S3. every hole adds testing sample 50 L, if negative control, positive control (what positive control of negative control is that test kit carries) each 2 holes, every hole adds negative control (or positive control) each 50 L, and sets blank 1 hole；
S4. every hole adds 50 L enzyme conjugates (blank control wells is not added with), fully mixes, shrouding, is placed in 37 DEG C and hatches 30min；
S5. wash plate: discard liquid in hole, fill each hole with the working concentration cleaning mixture of step S1 configuration, stand 5s, dry, pat dry after being repeated 5 times；
S6. every hole adds each 50 L of developer A, developer B, mixing, shrouding, is placed in 37 DEG C and hatches 15min；
S7. every hole adds stop buffer 50 L, mixing；Microplate reader reading, wavelength is 450nm and 630nm dual wavelength, first returns to zero by blank well, then reads each hole OD value.
Result judges: COV=negative control mean OD value × 2.1(negative control OD value, is calculated by actual OD value higher than 0.050 by 0.050 calculating less than 0.050).As sample to be tested OD value >=COV, it is judged that for the positive；When sample to be tested OD value is < during COV, it is judged that for feminine gender.
3, qualification result
8 F0 are for there being positive gomphosis mouse 4 in mice, Chi-meric mice positive rate is 50%.
Experimental example
3
Embodiment
1
Preparation
1.3
Copy
C
Type
HBV
Transgenic mice pass on qualification
1, the Chi-meric mice (F0 generation) embodiment prepared and normal KM Mus mate, and obtain F1 generation mice 32.
2, the statistics of mouse chimera rate, and ELISA detects the method for the hepatitis B surface antigen (HBsAg) in serum of transgenic mice and urine and hepatitis B virus e antigen (HBeAg) with embodiment 2.
3, qualification result
Chi-meric mice positive rate is 15.6% for positive gomphosis mouse 5 in (1) 32 F1 generation mice, shows that the inventive method builds the Chi-meric mice obtaining passing on.
(2) 5 F1 generation gomphosis mouse numbered B1～B5 respectively.
The result of ELISA detection hepatitis B surface antigen (HBsAg) and hepatitis B virus e antigen (HBeAg) shows, HBsAg and HBeAg in B1～B5 mice serum and urine is the positive.Record OD value as shown in table 1:
HBsAg and HBeAg in table 1 ELISA detection F1 generation gomphosis mouse serum and urine
Comparative example
1
The method of pronuclear microinjection is prepared
C
Type
HBV
Transgenic mice
1, the method for pronuclear microinjection prepares c-type HBV transgenic mice
(1) injection HBV(C is first prepared) DNA fragmentation.
(2) 6～8 week old well-developed FVB/N dams is selected, pregnant mare serum gonadotrop(h)in (PMSG) PMSG(10U) lumbar injection, human chorionic gonadotropin HCG(10U is injected) after 48h, hormone induction ovulation is mated by 1:1 with FVB/N public affairs Mus after processing, the cloudy bolt positive of m seq inspection as microinjection with germ cell donor Mus.Ligation KM public affairs Mus is mated with normal KM dams, chooses and have cloudy bolt and vaginal orifice swelling, ruddy dams as the receptor Mus of zygote transplation after injection.By HBV(C) DNA solution utilizes pronuclear microinjection method to be directly injected in the protokaryon of the unicellular germ cell of FVB/N that super row obtains, and takes the zygote transplation of injection survival in the fallopian tube of pseudo-fetus KM dams.After its conceived farrowing, carry out PCR detection.
Referring specifically to (2011) articles such as Chen Meijuan " preparation of 1.3 copy C genotype HBV transgenic mices and detection ".
2, Comparative result
(1) compared to the method for procaryotic DNA microinjection, transgenic mice prepared by blastaea of the present invention injection ES cell directly can judge the chimeric rate of mice by hair color, and without carrying out PCR detection.
(2) method using procaryotic DNA microinjection, gene is random integration, and the PCR positive rate in the c-type transgenic mice F0 generation obtained is 10.3%.
And great many of experiments shows, the mouse chimera rate that the present invention is obtained by blastaea injection ES cell has 40%～60%, substantially increases the transgene efficiency of mice.It addition, the method that the present invention uses blastaea injection ES cell, HBV-C gene can pinpoint restructuring ES cell, and the c-type HBV allophenic mice positive rate obtained is higher, and positive rate is 50%, hence it is evident that the c-type HBV allophenic mice obtained higher than prior art.
(3) as described above, the method for procaryotic DNA microinjection builds the serum of transgenic mice and urine HBsAg obtained and HBeAg is difficult to detect, serum quantitative fluorescent PCR displays that HBV DNA is low duplication expression, and copy number is 102～103Copy/mL.
HBsAg and HBeAg in the blastaea of the present invention injection serum of transgenic mice prepared of ES cell and urine is the positive, and can high efficient expression, data are as described in Example 3.
SEQUENCE LISTING
<110>Hospital No.458 of P.L.A.
The construction method of<120>1.3 copy C genotype HBV transgenic mices
<130>
<160> 1
<170> PatentIn version 3.2
<210> 1
<211> 4437
<212> DNA
<213>1.3 extraordinarily long segment c-type HBV plasmids
<400> 1
actagttgtg ctgccaactg gatcctgcgc gggacgtcct ttgtctacgt
cccgtcggcg 60
ctgaatcccg cggacgaccc gtctcggggc cgtttgggac tctaccgtcc
ccttcttcat 120
ctgccgttcc ggccgaccac ggggcgcacc tctctttacg cggtctcccc
gtctgtgcct 180
tctcatctgc cggaccgtgt gcacttcgct tcacctctgc acgtcgcatg gagaccaccg
240
tgaacgccca ccaggtcttg cccaaggtct tacataagag gactcttgga
ctctcagcaa 300
tgtcaacgac cgaccttgag gcatacttca aagactgttt gtttaaggac
tgggaggagt 360
tgggggagga gattaggtta aaggtctttg tactaggagg ctgtaggcat
aaattggtct 420
gttcaccagc accatgcaac tttttcacct ctgcctaatc atctcatgtt
catgtcctac 480
tgttcaagcc tccaagctgt gccttgggtg gctttggggc atggacattg
acccgtataa 540
agaatttgga gcttctgtgg agttactctc ttttttgcct tctgacttct
ttccttctat 600
tcgagatctc ctcgacaccg cctcagctct gtatcgggag gccttagagt
ctccggaaca 660
ttgttcacct caccatacag cactcaggca agctattctg tgttggggtg
agttgatgaa 720
tctggccacc tgggtgggaa gtaatttgga agacccagca tccagggaat
tagtagtcag 780
ctatgtcaat gttaatatgg gcctaaaaat cagacaacta ttgtggtttc
acatttcctg 840
tcttactttt ggaagagaaa ctgttcttga gtatttggtg tcttttggag
tgtggattcg 900
cactcctcct gcttacagac caccaaatgc ccctatctta tcaacacttc
cggaaactac 960
tgttgttaga cgacgaggca ggtcccctag aagaagaact ccctcgcctc
gcagacgaag 1020
gtctcaatcg ccgcgtcgca gaagatctca atctcgggaa tctcaatgtt
agtatccctt 1080
ggactcataa ggtgggaaac tttactgggc tttattcttc tactgtacct
gtctttaatc 1140
ctgagtggaa aactccctct tttcctcaca ttcatttaca ggaggacatt
attaatagat 1200
gtcaacaata tgtgggccct cttacagtta atgagaaaag gagattaaaa
ttaattatgc 1260
ctgctaggtt ctatcctaac cttaccaaat atttgccctt ggacaaaggc
attaaacctt 1320
attatcctga acatgcagtt aatcattact tcaaaactag gcattattta
catactctgt 1380
ggaaggctgg cattctatat aagagagaaa ctacacgcag cgcctcattt
tgtgggtcac 1440
catattcttg ggaacaagag ctacagcatg ggaggttggt cttccaaacc
tcgacaaggc 1500
atggggacaa atctttctat tcccaatcct ctgggattct ttcccgatca
ccagttggat 1560
cctgcgttcg gagccaactc aaacaatcca gattgggact tcaaccccaa
caaggatcat 1620
tggccagagg caaatcaggt aggagcggga gcattcgggc cagggttcac cccacctcac
1680
ggcggtcttt tggggtggag ccctcaggct cagggcatgt tgacaactgt
gccagtagca 1740
cctcctcctg cctccaccaa tcggcagtca ggaagacagc ctactcccat
atctccacct 1800
ctaagagaca gtcatcctca ggccatgcag tggaagaatt ccacaacatt
ccaccaaact 1860
ctgctagacc ccagagtgag gggcctatat tttcctgctg gtggctccag
ttccggaaca 1920
gtaaaccctg ttccgactac tgcctcaccc atatcgtcaa tcttctcgag
gactggggac 1980
cctgcaccga acatggagag cacaacatca ggattcctag gacccctgct
cgtgttacag 2040
gcggggtttt tcttgttgac aagaatcctc acaataccgc agagtctaga
ctcgtggtgg 2100
acttctctca attttctagg gggagcaccc acgtgtcctg gccaaaattc
gcagtcccca 2160
acctccaatc actcaccaac ctcttgtcct ccaatttgtc ctggctatcg
ctggatgtgt 2220
ctgcggcgtt ttatcatatt cctcttcatc ctgctgctat gcctcatctt
cttgttggtt 2280
cttctggact accaaggtat gttgcccgtt tgtcctctac ttccaggaac
atcaactacc 2340
agcacggggc catgcaagac ctgcacgatt cctgctcaag gaacctctat
gtttccctct 2400
tgttgctgta caaaaccttc ggacggaaac tgcacttgta ttcccatccc
atcatcctgg 2460
gctttcgcaa gattcctatg ggagtgggcc tcagtccgtt tctcctggct
cagtttacta 2520
gtgccatttg ttcagtggtt cgtagggctt tcccccactg tttggctttc
agttatatgg 2580
atgatgtggt attgggggcc aagtctgtac aacatcttga gtcccttttt
acctctatta 2640
ccaattttct tttgtctttg ggtatacatt tgaaccctaa taaaaccaaa cgttggggct
2700
actcccttaa cttcatggga tatgtaattg gatgttgggg tactttacca
caagaacata 2760
ttgtactaaa aatcaagcaa tgttttagaa aactgcctgt aaatagacct
attgattgga 2820
aagtatgtca aagaattgtg ggtcttttgg gctttgctgc cccttttaca
caatgtggat 2880
atcctgcctt aatgccttta tatgcgtgta tacaatctaa gcaggctttc
actttctcgc 2940
caacttacaa ggcctttctg tgtaaacaat atctgaacct ttaccccgtt
gcccggcaac 3000
ggtcaggtct ctgccaagtg tttgctgacg caacccccac tggatggggc
ttggctattg 3060
gccaccgccg catgcgtgga acctttgtgg ctcctctgcc gatccatact
gcggaactcc 3120
tagcagcttg ttttgctcgc agccggtcag gggcgaaact catcggaacc
gacaactctg 3180
ttgtcctctc tcgcaaatac acctcctttc catggctgct agggtgtgct
gccaactgga 3240
tcctgcgcgg gacgtccttt gtctacgtcc cgtcggcgct gaatcccgcg
gacgacccgt 3300
ctcggggccg tttgggactc taccgtcccc ttcttcatct gccgttccgg ccgaccacgg
3360
ggcgcacctc tctttacgcg gtctccccgt ctgtgccttc tcatctgccg
gaccgtgtgc 3420
acttcgcttc acctctgcac gtcgcatgga gaccaccgtg aacgcccacc
aggtcttgcc 3480
caaggtctta cataagagga ctcttggact ctcagcaatg tcaacgaccg
accttgaggc 3540
atacttcaaa gactgtttgt ttaaggactg ggaggagttg ggggaggaga
ttaggttaaa 3600
ggtctttgta ctaggaggct gtaggcataa attggtctgt tcaccagcac
catgcaactt 3660
tttcacctct gcctaatcat ctcatgttca tgtcctactg ttcaagcctc
caagctgtgc 3720
cttgggtggc tttggggcat ggacattgac ccgtataaag aatttggagc
ttctgtggag 3780
ttactctctt ttttgccttc tgacttcttt ccttctattc gagatctcct
cgacaccgcc 3840
tcagctctgt atcgggaggc cttagagtct ccggaacatt gttcacctca
ccatacagca 3900
ctcaggcaag ctattctgtg ttggggtgag ttgatgaatc tggccacctg
ggtgggaagt 3960
aatttggaag acccagcatc cagggaatta gtagtcagct atgtcaatgt
taatatgggc 4020
ctaaaaatca gacaactatt gtggtttcac atttcctgtc ttacttttgg
aagagaaact 4080
gttcttgagt atttggtgtc ttttggagtg tggattcgca ctcctcctgc ttacagacca
4140
ccaaatgccc ctatcttatc aacacttccg gaaactactg ttgttagacg
acgaggcagg 4200
tcccctagaa gaagaactcc ctcgcctcgc agacgaaggt ctcaatcgcc
gcgtcgcaga 4260
agatctcaat ctcgggaatc tcaatgttag tatcccttgg actcataagg
tgggaaactt 4320
tactgggctt tattcttcta ctgtacctgt ctttaatcct gagtggaaaa
ctccctcttt 4380
tcctcacatt catttacagg aggacattat taatagatgt caacaatatg
tgggccc 4437
Claims (4)
1. the construction method of a copy C genotype HBV transgenic mice, it is characterised in that step is as follows:
S1. the foundation of embryonic stem cell line；
S2. 1.3 extraordinarily long segment c-type HBV plasmid electricity are transduceed to embryonic stem cell, through identifying the embryonic stem cell obtaining the positive, be enlarged cultivating；
S3. in positive embryos stem cell injection to blastaea, the blastaea injected is transplanted to the uterus of pseudo-fetus dams, cultivates and obtain 1.3 copy C genotype HBV transgenic mices；
Wherein, described in step S2, the method for electricity transduction is as follows:
S21. it is individual cells by the embryonic stem cell line trypsinization of cultivation, adds and terminate culture medium and terminate digestion, centrifugal, remove supernatant, then suspend with subtracting blood serum medium opti-MEM；
S22., suspension adds the c-type HBV plasmid needed for electricity transduction, mixing, transfers in electricity revolving cup, carry out electricity transduction with electroporation, be rapidly added cell culture medium after electricity transduction, be then taped against in cell plates；
S23. second day, replace medium to the culture medium of puromycin-resistant, carry out every day changing liquid；
S24., after cultivating 5～7 days, the cell only having proceeded to plasmid just can grow into colony clone, and what picking screened respectively is cloned in cell plates, 1, each hole cell clone；
S25. second day, by passage to new cell plates, after again covering with, cell was used for extracting DNA and carries out PCR qualification, obtains positive colony；
S26. it is enlarged the cell of the positive colony obtained cultivating, obtains positive embryos stem line；
Wherein, described embryonic stem cell is mouse embryo stem cell, and described embryonic stem cell line is mice embryonic stem cell system.
Construction method the most according to claim 1, it is characterised in that the method for building up of embryonic stem cell line described in S1 is as follows:
S11. taking the female pregnant Mus of C57BL/6J of conceived 3.5 days, disconnected neck is put to death；
S12. the pregnant Mus sterilization with ethanol, S11 put to death；
S13. open pregnant Mus abdominal cavity, repair removing fat and mesentery, cut bilateral uterine from fallopian tube root and lower end, uterus crotch, uterus bicornis is cut off at vagina；
S14. syringe sucks embryo washing water, and the syringe needle of syringe is cut needle point before the use and polished sterilizing；By syringe in one end intron uterine cavity in uterus, clamping, it is blown into embryo washing water, then the other end from uterus inserts syringe, is blown into embryo washing water；
S15. being taken out by above-mentioned embryo washing water and be placed in plate, plate is placed under anatomical lens, holds pipe in the month with mouth and draws a small amount of embryo washing water, by blastaea sucking-off；
The most then blastaea is put in embryo culture medium, observe, select reasonable blastaea；
S17. the blastaea of selection is blown into the cell plates being vaccinated with feeder layer cells in advance, uses Establishing Mouse Embryonic Stem Cell Line culture medium instead, every hole is put 1 blastaea；
S18. putting into incubator to cultivate, blastaea is gradually grown up, the most adherent, grows germinal layer and launches, and the inner cell of blastaea is constantly bred；
S19. cultivating after 4～6 days, the inner cell of blastaea becomes a reunion cylindricality or protruding cell mass, now can the inner cell mass of discrete blastaea, obtain embryonic stem cell line.
Construction method the most according to claim 1, it is characterised in that described in S3, the method for injection is as follows:
S31. the positive embryos stem line that electricity transduction obtains is digested resuspended one-tenth individual cells；
S32. punching on the zona pellucida of blastaea with laser rupture of membranes instrument, get to inside segmentation cavity with entry needle by positive embryos stem cell, after then cultivating 2～3 hours by KSOMaa embryo culture medium, basis of microscopic observation blastaea form is recovered.
Construction method the most according to claim 1, it is characterized in that, pseudo-fetus dams described in S3 obtains by the following method: KM dams and KM are ligatured public Mus and carries out mating raising according to female-male proportion 2:1, select cloudy bolt and vaginal orifice swelling, ruddy dams secondary morning, it is pseudo-fetus dams, it is denoted as 0.5 day pseudo-fetus dams, and blastaea is transplanted in 2.5 days pseudo-fetus dams.
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| Title |
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| 1.3拷贝C基因型HBV转基因小鼠的制备及检测;陈媚娟 等;《解放军医学杂志》;20110901;第36卷(第9期);第967页摘要，第967-968页第1.2节,第967页左栏第1段 * |
| 3copy C 基因型HBV转基因小鼠的制备;陈媚娟 等;《中国比较医学杂志》;20101031;第20卷(第10期);第74页 * |
| 囊胚注射转基因ES细胞制作嵌合体的研究;付志新 等;《畜牧兽医学报》;20071231;第38卷(第2期);第149页摘要，第151页1.10节 * |
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