WO2012113124A1 - New method for improving transgenic efficiency in animals - Google Patents

Abstract

A new method for improving transgenic efficiency in animals comprises transfecting the germ cell lines of male and female mice respectively with a plasmid carrying an exogenous gene by multi-point injection before the male and female mice are bred, and then male mice 1-5 days and 35-40 days after surgical operation and female mice 1-7 days after surgical operation are selected and bred, to obtain transgenic mice offspring. In the transgenic method, with sperm and oocyte as vectors, the exogenous gene is respectively introduced into the sperm and the oocyte through method improvement, and the exogenous gene carried by the sperm and the oocyte is further integrated in embryo through the fertilization process, thereby improving the introduction efficiency of the exogenous gene into the cell nucleus, and express the exogenous gene with a high efficiency. The method provides a transgenic system mediated by the male and female germ cells in combination, which can improve the transgenic efficiency in large mammals, thereby providing a new way for breeding livestock.

Description

 A new method for improving the efficiency of transgenic animals

TECHNICAL FIELD This invention relates to methods of transgenic. Specifically, the present invention relates to a transgenic method using sperm and oocytes as a vector, introducing a foreign gene into a sperm and an oocyte, respectively, and further integrating the foreign gene carried by the two in the embryo through a fertilization process, Increase the efficiency of introduction of foreign genes into the nucleus.

Background Art Transgenic animals are experimentally introduced into a fertilized egg of an animal, which is proliferated and stably inherited to the offspring. Scientists have successfully obtained transgenic mice, rabbits, chickens, goats, pigs, sheep, cattle, frogs and a variety of genetically modified fish. Transgenic animals are a new milestone in the development of genetics. They show great advantages and broad application prospects in gene function and expression research, establishment of human disease models, supply of organ transplant materials, precious pharmaceutical protein production, and animal husbandry.

[Zeng Yitao. Transgenic Animals and Biomedical Industry. Bulletin of Biology, 1999, 34 (4) : 1-3. Wang Jianchen, Ma Baohua. Prospects of Transgenic Technology in Animal Husbandry and Veterinary. Advances in Animal Medicine, 2000, 21 (1) : 1-8. ]. In the past three decades of the development of transgenic technology, people have explored a variety of methods. Nowadays, people are increasingly demanding GM methods. In addition to high efficiency, simple operation, and more hope that the introduction method is safe and reliable, it is best to introduce or transform multiple genes according to people's wishes. The sperm carrier method has been confirmed by researchers after more than 10 years of controversy, which is a relatively simple and effective transgenic method. At the same time, oocytes, as the most important carrier of genetic material, have received widespread attention from the beginning.

The spermatogonial stem cells have self-proliferation ability, and can produce continuously updated stem cells and differentiated spermatogonia cells, two cells formed by each division, one developed by primary spermatocytes, secondary spermatocytes, sperm cells, and the like. It is a sperm; a spermatogonial stem cell that remains in its original undifferentiated state and continues to divide and proliferate, so that after transfection of spermatogonial stem cells, the animal can produce transfected sperm more consistently. Due to this superiority, transfection of spermatogonial stem cells has also received much attention. Kuznetsov et al [Kuznetsov AV, Kuznetsova IV, Schit IY. DNA interaction with rabbit sperm cells And its transfer into ova in vitro and in vivo. Mol Reprod Dev, 2000, 56 : 292- 297. ] In the experiment, lentivirus and spermatogonial stem cell-mediated transfection of mice were used to obtain 40% transgenic mice. 6% of mice can pass on foreign genes to their offspring. As a transgenic method, the sperm carrier method is simple in operation, high in transgenic efficiency, and requires no special instruments. The more prominent effects are single-injection (ICSI) and sperm-mediated in vivo transfection. Due to the multi-linearization of the plasmid used in the sperm carrier method, the probability of destruction of the circular gene structure is high, and the introduced foreign gene is randomly integrated, so it is easy to cause damage or loss of the functional structure of the foreign gene. Live, thereby directly affecting or inhibiting its expression [Guo Zhiqin. Livestock Embryo Engineering. 1998, China Science and Technology Press.].

Studies have shown that most types of animal sperm have the ability to spontaneously bind to foreign DNA, with a binding rate ranging from % to 80. /. [Lavitrano M., Busnelli M., Cerrito MG, et al. Sperm-mediated gene transfer. Reprod Fertil Dev, 2006, 18 (1-2): 19-23.]. Spadaf ora [Spadaf ora C. Sperm cells and foreign DNA: a controversial relation. Bioessays, 1998, 20 : 955-964.] A hypothetical model of exogenous DNA internalization is proposed: normal injected sperm, acrosome posterior torso The ability of DNA-binding protein (DBP) to bind to foreign DNA is inhibited by IF-1 factor. When sperm is washed thoroughly or semi-serum-free semen is used, the inhibition of IF-1 is released and the foreign DNA interacts with DBP. The DNA-DBP complex, the DNA-DBP complex binds to CD4 and assembles into a DNA-DBP-CD4 complex. The complex internalization reaches the nuclear matrix through the nucleus, and the foreign DNA is dissociated in the nuclear matrix region and is in close contact with the chromosomal DNA of the sperm. The free protein complex circulates to the membrane and transports new DNA molecules. The integration region of exogenous DNA in the sperm genome mostly occurs near the recognition sequence of the nuclear matrix region or topoisomerase ;; while the integration time occurs mostly before and after fertilization, there is a single-strand break on the chromosomal DNA of the male and female pronucleus of the fertilized egg. Gap, early embryonic cells actively divide and proliferate, DNA replication process leaves a large number of replication forks and replication fragments, these single-stranded gaps provide the premise and guarantee for the integration of foreign DNA into the genome. In addition, exogenous DNA activates the ribozyme system and DNA repair system as it enters the host cell. The ribozyme also cleaves the chromosomal DNA while degrading the foreign DNA. Under the action of the DNA repair system, the foreign DNA is integrated into the genomic DNA. The integration of foreign DNA in the host genome differs depending on the type of offspring produced during the period of occurrence. If the foreign DNA is integrated into the host genome during the pronuclear stage of the fertilized egg or in the I cell stage of the embryo, the transgenic animal is obtained, and the animals that are recombined after the embryonic cell stage are chimeras, and those that fail to integrate into the genome. Exogenous DNA is gradually degraded by ribozymes in embryonic cells [Chan AW, Luet jens CM, Schatten GP Sperm-mediated gene transfer. Curr Top Dev Biol, 2000, 50: 89-102.].

 At present, the use of sperm as a vector to mediate gene transfer to obtain transgenic animals is mainly achieved by the following methods: in vitro sperm transfection and in vivo sperm transfection. In vitro sperm transfection can be further divided into sperm and exogenous DNA direct co-incubation, in vitro electroporation and liposome-mediated transfection; in vivo sperm transfection can be divided into intra-testicular injection, vas deferens Injection method, seminiferous tubule injection method. Testicular injection is the direct injection of exogenous DNA into the testes of male animals, transfection of sperm cells at various stages of development, transfection of sperm after in vivo maturation, natural or artificial insemination to obtain transgenic animals. Testicular injection is performed by Soto et al. [Sato M., Iwase R., Kasai K., et al. Direct injection of foreign DNA into mouse testis as a possible alternative of sperm-mediated gene transfer. Anim Biotech, 1994, 5 ( 1) : 19 - 31. ] was first used in 1994. The seminiferous tubules of animals such as pigs are surrounded by thick and opaque white membranes, and the liposome/DNA complex cannot be accurately injected into the curved fine tube, so the testicular injection is selected so that the foreign DNA can be effectively integrated into the vein. Male germ cells. 1999, Farre et al [Farre L., Rigau T., Mogas Τ., et al. Adenovirus-mediated introduction of DNA into pig sperm and offspring. Mol Reprod Dev, 1999, 53 (2): 149- 158. In pig testis transfection, 47% of sperm transfection positive and 22% of pig embryo expression positive rate, the same year Sato et al [Sato M. Testis-mediated gene transfer (TMGT) in mice : successful transmission of introduced DNA from F0 to Transgenics, 1999, 3: 11-22. Transgenic mice were obtained by intratesticular injection. Giui li [42] and the like by this method enable the stable expression of foreign genes in sperm. Xiao Hongwei et al [Xiao Hongwei, Zheng Xinmin, Chen Sihuai et al. Sperm-mediated production of hCD59 gene pig. Journal of Huazhong Agricultural University, 2006, 25 (2): 170- 173.] To study the production of transgenic pigs for xenotransplantation by sperm-mediated method For the efficiency, the recombinant plasmid pcDNA3-hCD59 was injected into the testis of pigs by dot injection to obtain PCR positive pigs (positive rate 4.3%).

Mammals form a certain number of oocytes before and after the birth of a fetus. During the fetus, there are a considerable number of oogonia cells formed by primordial germ cells in the ovary. The oogonia cells further form primary oocytes (this phase is also known as the GV phase), at which time the oocyte growth is extremely insignificant and can last from several months to several decades. After sexual maturity, the oocyte enters a rapid growth stage, forming a large amount of egg yolk, accumulating various nutrients, completing the differentiation of the egg, synthesizing and storing the developmental information required for early embryo development, and completing the first mature division from the ovary. Entering the fallopian tube becomes a secondary oocyte, then quickly enters the second mature division, and terminates in the second meiotic mid-term waiting for fertilization. If it is not within 24 hours after ovulation Fine, secondary oocytes will degenerate; if the sperm meets fertilization, the secondary oocytes complete the second mature division, the oocytes are activated by sperm, mainly in the efflux of the egg cortex particles, meiosis The recovery and discharge of the second polar body, the formation of the male and female pronucleus. The maturation of the oocyte is divided into the maturation of the nucleus and the maturation of the cytoplasm. At present, regarding the maturity of the cytoplasm, the development status of the blastocyst in the later stage and the health status of the offspring are the sole criteria for evaluation.

Since the establishment of spermatogonia cell transplantation technology for the first time [Hofmann MC, Laura BS, Luis D., et al. Immortalization of Mouse Germ Line Stem Cells. Stem Cells, 2005, 23 (2): 200-210.] , from the same species to the heterogeneous have been successfully reported. Oocytes, also as a totipotent cell, can undergo cell differentiation and enter embryonic development, and can extend all the genetic information stored in the genome to all cells in the organism. Transgenic methods are used to transfect foreign genes into oocytes. If foreign genes are integrated into the genome, all transgenic positive cells can be used for transgenic animal production. Oocytes play a very important role in the development of transgenic technology. As early as 1980, the first attempted transgenic method, pronuclear microinjection, used oocytes as a material for microinjection. This is not only because the oocyte is the largest cell in the animal's body, but also because it plays an irreplaceable role in animal reproduction and inheritance. In the 1990s, this technology was commercialized, and some companies could even provide transgenic animals that integrated foreign genes, but the efficiency was still too low, and only 4% of oocytes could continue to develop after injection. When Anthony et al. used a lentivirus to transfect bovine oocytes, [Chan AW, Homan EJ, Ballou LU, et al. Transgenic cattle produced by leverse-transcribed gene transfer in oocytes. Proc Nail Acad Sea USA, 1998, 95 (24 ): 14028-14033. ] , At the end of the second mitosis of the oocyte, the nuclear membrane will rupture for a short time. The foreign gene is successfully mediated by lentivirus and enters the nucleus of the oocyte during this period. This opens up a new way for people to use oocytes as a transgenic vector, and the strong barrier of the oocyte nuclear membrane can be broken. Cabot et al. cultured porcine oocytes to maturity in vitro [Cabot RC, Kuhholzer B., Chan AW, et al. Transgenic pigs produced using oocytes infected with a retroviral vector. Anim Biotech, 2001, 12 (2): 205-214 . . , transfecting green fluorescent protein with retrovirus as a vector, followed by in vitro fertilization, and transplanting the fertilized egg to the recipient sow. Two litters of piglets were obtained, and one piglet was randomly selected from each litter to detect the foreign gene was positive. The success of this approach suggests that the probability of direct transfection of oocytes to obtain transgenic offspring is significant. Scientists try to digest mature oocytes and mix them with DNA plasmids and liposomes to obtain transgenic mice. [Wu Desheng, Wang Xiaoyun, Wu Congmei et al. Liposome-mediated exogenous DNA transfection of gold hamster oocytes in vitro, carcinogenesis. Distortion. Mutation. 2004, 16 (3): 137-141.]. This is the first time in China that the golden hamster oocytes can be transfected with liposome-encapsulated foreign DNA, that is, golden hamster oocytes can be used as a carrier for foreign genes. This new pathway is to study foreign genes. Expression in golden hamster oocytes provides an experimental basis. Zhang Qingjian et al [Zhang Qingjian, Huang Tianhua, Xie Qingdong et al. Transfection of HBV DNA recombinant plasmid into mouse oocytes. Carcinogenesis. Distortion. Mutation. 2004, 16 (6) : 324- 327.] This method established HBV vertical infection. Mouse model. It was confirmed that the mouse oocyte zona pellucida did not completely block HBV from entering the oocyte. The ability to absorb foreign DNA with the participation of liposomes. The zona pellucida is a non-cellular structure in which the outer layer of the egg is mainly composed of glycoproteins. It protects fragile egg cells and embryos from external damage. Grabarek et al [Joanna B. Grabarek, Berenika Plusa, David M. Glover et al. Efficient delivery of dsRNA into zona-enclosed mouse oocytes and pre implant at ion Embryos by electroporation. Genesis, 2002, 32 (4): 269-276. ] It is proved that the obstruction effect of the transparent belt can be weakened by the electroporation method. Laurema et al [Laurema A., Heikkila A., Keski Nisula L., et al. Transfection of oocytes and other types of ovarian cells in rabbits after direct injection into uterirn arteries of adenoviruses and plasmid liposomes. Gene Ther, 2003, 10 (7) : 580-584. ] When the ovary was transfected with a mixture of adenovirus or DNA plasmid liposomes, it was found that the primordial germ cell nuclear primary oocytes without zona pellucida were susceptible to zona pellucida protection. Secondary oocytes are not easily transfected, indicating that the zona pellucida blocks the entry of foreign DNA.

Liposomes can be used to transfer a variety of substances into many types of cells in animals, plants and microorganisms. Liposomes can carry gene fragments of different sizes, plasmid DNA, etc. The encapsulation of liposomes can prevent the degradation of nucleases and prevent DNA from being diluted, which can increase the transfection rate. The main forms of action of liposomes as nucleic acid carriers and cells include intermembrane transport, contact release, adsorption, fusion and endocytosis [Xiao HZ, Huang L. DNA transfection mediated by cationic liposomes containing lipopolylysine: characterization and machanism of action BBA, 1994, 1189 : 195-203. ] c Lipofectamine-2000 is a highly efficient and stable cationic liposome transfection reagent. Its mechanism of action is that the positively charged liposome forms a liposome/DNA complex with the negatively charged phosphate group of the nucleic acid. This complex still has a positive charge due to the liposome located on the outer layer, and it is associated with the cell membrane. The negative charge of the sialic acid residue on the complex combines with the cell to be absorbed by the cell, allowing the DNA to enter the cell. The application of transgenic technology in mammalian gene expression has become one of the most significant advances in experimental biology and applied biology for nearly 30 years. Since the establishment of GM technology, scholars from all over the world have been striving to seek technical improvements to simplify the operation and increase the positive rate of transgenic animals. In recent years, the use of sperm as a carrier to produce transgenic animals has become a research hotspot, but the positive rate of transgenic animals produced by this method varies greatly, ranging from 4% to 90%. Therefore, verification and optimization of this method is imperative. . Due to the strong barrier of the oocyte zona pellucida, there is a conservative attitude towards the use of oocytes as a transgenic vector. However, if the oocyte is a provider of half of the genetic material of the offspring, if it can be successfully transfected, it will undoubtedly increase the positive rate of transgenes in the offspring. Therefore, the present invention uses simultaneous transfection of sperm and oocytes, and then allows individuals carrying transgenic positive sperm and oocytes to naturally mate to produce transgenic offspring, hoping to obtain higher transgenic efficiency by this method, this method will be The use of this method for transgenic studies in other animals can provide valuable references, especially for the application of large livestock oocyte-mediated methods. The method of the invention is convenient to operate, does not require expensive equipment, and has low technical requirements for operators. The invention obtains efficient and stable transfection results, is beneficial to the promotion of animal transgenic technology, further improves breeding efficiency, produces bioreactors with low cost, provides cheap xenotransplant organs, and cultivates new varieties of transgenic animals.

SUMMARY OF THE INVENTION The present invention discloses a novel method for improving the efficiency of transgenic animals, comprising the following steps:

 1) preparing a plasmid with a foreign gene;

 2) introducing a plasmid carrying a foreign gene into a testis tissue of a male animal by spot injection;

3) introducing a plasmid carrying the foreign gene into the ovarian tissue of the female animal by spot injection;

4) Male testicular male animals are mated with female animals to obtain progeny of transgenic animals. The dot injection method of the present invention directly injects a foreign gene into the testis and ovarian tissue of an animal. The animal of the present invention is a mammal.

 The method of the present invention can be used to increase the positive rate of foreign genes carried by progeny animals.

 The invention also discloses a novel method for improving the transgenic efficiency of a mouse, comprising the following steps:

1) preparing a plasmid with a foreign gene;

2) introducing a plasmid carrying the foreign gene into the testis of the male mouse by spot injection; 3) introducing a plasmid carrying the foreign gene into the ovary of the female mouse by spot injection;

 4) Pick out the male rats 1-5 days and 35~40 days after the operation of the spot injection method, and inject the female hand for 1-7 days after the injection;

 5) The selected male mouse and the female mouse are mated to obtain progeny of the transgenic mouse.

 The present invention utilizes the method to initially carry out transgenic research on large mammalian sheep, and the results show that the method of the present invention can improve the transgenic efficiency of large mammals and provide a new way for animal husbandry breeding.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a surgical drawing of a male mouse. Figure A shows the pre-operative, Figure B shows the testes of the male rats, Figure C shows the photos after the unilateral testicular injection, and Figure D shows the photos after the double-sided injection.

Figure 2 Surgical diagram of the mother. Figure A shows the ovary of the mother exposed by surgery, and Figure B shows the ovary injected with a micro-injector. Figure C shows that the ovary is blue and has no spill after injection.

Figure 3 is a double-digested plasmid map. The plasmid pIRES-eGFP was digested with the restriction enzymes BanHI and Xhol to obtain a 1060 bp gene fragment.

Figure 4 shows the electropherogram of the amplification results. The bright band is a 298 bp gene of interest.

Figure 5 PCR detection of the positive rate of exogenous DNA in testis and semen. The positive rate in semen is still at a high level at 35-40 days. Figure 6. Detection of PCR positive rate in ovarian tissue and follicular fluid. The positive rate showed a downward trend within 1-5 days after the ovarian management of the mother.

Figure 7 Observation of oocytes under a fluorescence microscope. The results showed that the oocyte transgenic effect was good. Figure 8. Electrophoresis pattern of PCR amplification of F1 generation mouse genomic DNA. There was a distinct bright band at 300 bp, which was the same size as the positive control, but the tail of the control group did not amplify the band.

Figure 9. RT-PCR electropherogram of each organ. Lane 1 is a negative control; Lane 2 is mouse heart tissue; Lane 3 is tail tip tissue; Lane 4 is mouse kidney tissue; Lane 5 is mouse muscle tissue; Lane 6 is mouse liver tissue; Lane 7 is small Mouse testis tissue; Lane 8 is mouse ovarian tissue.

Figure 10. Maternal dot injection method for obtaining transgenic efficiency maps of offspring mice. Among them, the progeny of the testicular-injected male and the normal female are called the A group, and the ovarian-injected female and the normal male are mated. In the group B, the testes of the male rats and the ovaries of the mother rats were operated, and the progeny obtained from mating were called group C. Figure 11 shows the transgenic efficiency map of offspring sheep by female dot injection method. Among them, group A is the gene positive rate of progeny obtained by mating male testicular male sheep with normal female sheep, and group B is the positive rate of progeny of maternal ovarian-injected female sheep mating with normal male sheep, and group C is performing management. The positive rate of the progeny of the male sheep treated with the injection method and the female sheep.

Example 1 Female generation of injection injection to obtain transgenic offspring mice

I. Experimental materials and reagent preparation

 Experimental material

Kunming white mice, 5-8 weeks old, were purchased from the Experimental Animal Center of the Chinese Academy of Medical Sciences. A total of 180 female rats and 90 male rats were used in the experiment. Trypan blue dye solution, sodium pentobarbital, PMSG, HCG, human penicillin/streptomycin, tryptone, yeast extract, DEPC (diethyl pyrocarbonate), maleic acid, Tween-20, SDS, Agar powder, DH5ct competent cells, Lipofectin2000, 2XTaq PCR MasterMix, X-gal, IPTG, One-Step RT Kit, DIG High Prime DNA Labeling and Detection Starter Kit 1, agarose gel recovery kit, tissue genomic DNA extraction reagent Box; reinforced fluorescent protein granule: pIRES-eGFP, endotoxin-free plasmid, endonuclease BamH I, endonuclease Xho I, M2 medium.

 2. Reagent preparation

1) Saline: 0.9% NaCl solution, weigh 9g NaCl, dilute to 1000mL with ultrapure water, autoclave.

2) Hormone: Take one of PMSG and HCG (1000 IU) dissolved in 1 mL of normal saline or ultrapure water at a concentration of 1000 IU · mL, dispensed in 0.2 mL sterile centrifuge tube, and stored at -20 °C. Use once after thawing.

3) Cleaning solution (commonly used, sulfuric acid concentration is 25%): 1000 g of potassium dichromate, 2500 mL of concentrated sulfuric acid, and 7500 mL of double distilled water.

 4) 0.1% DEPC treated water: Add 1 mL of DEPC in 1000 mL of double distilled water, mix by shaking, sterilize at room temperature overnight, and store at 4 °C after use. (Note: The solutions related to embryo collection and RNA manipulation in the experiment were prepared with 0.1% DEPC treated water)

5) 50XTAE buffer: Tris base 242g, glacial acetic acid 57. lmL, 0.5 mol/L EDTA (pH 8.0) lOOmL, add water to lOOm

 6) LB liquid medium: Add 10 g of tryptone for bacterial culture, 5 g of yeast extract, 10 g of NaCl in 950 mL of distilled water, shake the container to complete dissolution and adjust the pH to 7.0 with 5 mol/L NaOH (about 0.2 mL). lOOOm autoclaved.

 LB solid medium: trypsin 10g, yeast powder 5g, NaCI 10g, 800 mL ddHzO dissolved, adjust the pH to pH 7.4 with 5mol / L NaOH (about 0.2mL), add 1.5% agar powder, autoclave, When the temperature of the medium is lowered to about 60 ° C, Ampicillin is added, and the plate is poured in an ultra-clean work. After cooling, the sealing film is sealed and stored at 4 ° C for use.

7) CaCl ₂ (lmol/L): Dissolve 54 g of CaCl ₂ · 63⁄40 in 200 mL of ultrapure water, filter and sterilize, or autoclave, dispense into 10 mL portions, and store at -20 °C.

8) Ampicillin sodium (100g/L) _: lg Ampicillin sodium is dissolved in 10mL of ultrapure water, filtered and sterilized, stored at -20 °C.

9) 1% agarose gel solution: Weigh agarose lg and add 100 mL of 1 XTBE solution and Goldene _W 5 μL.

 (Note: The buffer in the formulated lipogel solution should be the same as the concentration of the running buffer).

 10) PH8.3 Tris-boric acid - 3EDTA buffer: Weigh 10.78g Tris, 5.500g Boric acid 0.930GEDTA-Na2 Dissolve in deionized water, dilute to lOOmL, dilute 10 times when used.

 11) EB solution: Ethidium bromide (10mg/mL) is diluted 10 times.

 12) 0.5% trypan blue: Weigh 0.5g trypan blue and bring to a volume of 100mL.

 13) Sodium pentobarbital solution: Weigh 0.030 g of sodium pentobarbital and add 2 mL of normal saline. The concentration used is 0.015g/mL (currently available).

 14) Denaturant: 0.5 M NaOH; 1.5 M NaCl.

 15) Neutralizing solution: 1M Tris-HCl (pH 7.4); 1.5 M NaCl;

 16) 20XSSC: Dissolve 175.3g of sodium chloride and 88.2g of sodium citrate in 800mL of water, adjust the pH to 7.0 with sodium hydroxide, and add distilled water to 1000m for autoclaving.

 17) 10% SDS: Measure the electrophoresis grade SDSlOOg is dissolved in 900mL of distilled water, heated to 68 °C to help dissolve, add water to a volume of lOOOOmU

 18) Washing solution A: 2XSSC, 0.1% SDS.

 19) Washing liquid B: 0.1XSSC, 0.1% SDSo

20) Maleic acid solution: 11.608g maleic acid, 8.776g NaCl, 800mL water added After the mixture was dissolved, the pH was adjusted to 7.5 with solid NaHH, and the volume was adjusted to 1000 mL, and the mixture was autoclaved.

21) Washing liquid C: 11. 608g maleic acid, 8. 776g NaCl, add 800mL of water to stir and dissolve, adjust the pH to 7.5 with solid NaOH, dilute to 1000mL, autoclave after partitioning, cool to room temperature Add 3 mL of Tween-20 solution and mix.

 22) Balance buffer (Detection buffer): 12. l lg Tris, 5. 85g NaCl, add 800mL of water to stir and dissolve, adjust the value of 11 to 9.5 with concentrated hydrochloric acid (about 11^), to a volume of lOOOm after the high pressure Sterilize.

 23) Stopping solution (TE buffer): 10 mmol/L Tris-HCl, lmmol/L EDTA, adjusted to pH 8.0.

 24) Antibody solution: 2. 4 μΙ Anti-DIG + 12 mL l X Blocking solution.

25) Color-substrate Solution: 100 μΐ NBT/BCIP+ 5mL test solution (currently used, stored in the dark).

 26) Blocking solution: Dilute lO X Blocking Solution 10 times with maleic acid. 2. Experimental methods

1. Preparation of experimental rats Before the introduction of the mice, the rats were disinfected with a new clean, and the ultraviolet sterilization lamp was sterilized. After the introduction of the mouse, the breeding behavior of the male rats was trained to confirm that they had good reproductive performance. The mother can be used for experiments after it has been adapted for one week. The superovulation process was performed by intraperitoneal injection of pregnant horse serum gonadotropin (PMSG) 5 IU/only, and 48 h later, human chorionic gonadotropin (hCG) 5 IU/only.

 2. Extraction of RNA Vessels Glassware related to RNA manipulation was routinely washed, soaked with 0.1% DEPC at 37 °C overnight, autoclaved to remove DEPC, and then baked at 200 °C for 8 h. Plastic equipment and Eppendorf tubes and Tips use disposable products without RNAase. All solutions (except Tris) were added to DEPC to a final concentration of 0.1%, treated at 37 °C overnight, and then autoclaved. The Tris solution is prepared with treated diethylpyrocarbonate (DEPC) water, prepared and then autoclaved.

 3. Plasmid transformation

1) Competent cell DH5 α, transforming plasmid pIRES-eGFP.

Take 50 competent cells, place on ice, wait for it to thaw; add ly L plasmid, gently shake, and quickly ice bath for 30 min; 42 ° C water bath for 90 sec, 37 ° C water bath for 5 min; fast ice bath for 2 min, then add LB Mix the liquid medium to 1 mL, and incubate at 200 r/min for 60 min at 37 ° C in a shaker; take 500 μL of the above culture and apply it evenly on the solid LB medium plate containing ampicillin until the surface of the plate is completely liquid. After being absorbed, incubate at 37 ° C for 12 h.

 2) Pick the monoclonal cells into 10 mL LB sterile medium at 37 ° (shake overnight, and amplify them. The plasmid was extracted using a plasmid extraction kit, and then the purity and concentration were measured by an ultraviolet spectrophotometer.

 3) The plasmid extraction method is as follows according to the endotoxin-free plasmid extraction method:

a, 500 mL of bacterial liquid was poured into two 250 mL centrifuge cups, and centrifuged at 3500~5000 X g for 10 min at room temperature. b. Discard the supernatant, then add 7 mL of solution I / RNase A to the bacteria pellets and mix by pipetting with a 1 mL pipette.

c. Transfer the bacterial solution to a cleaned centrifuge tube that can withstand 12000 X g of 30 mL. Add 7 mL of Solution II, gently invert 7 to 10 times, and let stand for 5 to 10 minutes at room temperature.

d, add 10 mL of solution III, gently invert several times until white flocculent precipitate appears, leave it at room temperature for 5 min, centrifuge at room temperature 12000 X g for 10 min to remove genomic DNA and cell debris.

e. Take the supernatant, put it into a 50 mL DNA adsorption tube, centrifuge at 3000~4000 g for 5 min at room temperature, and discard the liquid below. After centrifugation for another 5 minutes, the following liquid was removed and the tube was used.

f. Add 5 mL of HB buffer to the tube just centrifuged, centrifuge at 3000~4000 X g for 5 min at room temperature, and remove the centrifuged liquid.

g. Add 10 mL of ethanol diluted DNA washing buffer, centrifuge at 3000~4000 X g for 5 min at room temperature, and remove the centrifuged liquid.

h, centrifuge with 10 mL of absolute ethanol 3000~4000 X g for 5 min at room temperature, discard the supernatant.

i, 3000~4000 X g empty tube centrifuge for 10 min, remove all ethanol.

j, vacuum drying 15 min ₀

k. Add an appropriate amount of double-distilled deionized water to 70 预 or TE buffer, leave it at room temperature for 2 min, and centrifuge at 5500 X g for 5 min to obtain plasmid DNA.

 4) The plasmid was subjected to double digestion with the endonucleases BamH I and Xho I, and the 1% agarose gel electrophoresis was used to determine whether the plasmid was the target plasmid. The plasmid concentration was determined by an ultraviolet spectrophotometer. The plasmid concentration was adjusted to lmg/mL and placed at _20 °C for use.

4. Probe labeling and DNA labeling 1) Design of PCR primers

 Based on the pIRES-eGFP plasmid sequence, a pair of primers was designed using the software Primer premier 5 to amplify the GFP fragment and label it as a probe. The upstream primer is 5'-TGCTTCAGCCGCTACCC-3,

(SEQ ID NO: 1), the downstream primer is 3, _AGT TCA CCT TGA TGC CGT TC-5, (SEQ ID NO: 2). The theoretical amplified fragment is 298 kb in length. The PCR primers were synthesized by Beijing Aoke Biotechnology Co., Ltd.

 2) PCR reaction system and conditions

 PCR reaction system:

 Template DNA 1 μL

 10 pmol/L PI 1 μL

 10 pmol/L P2 luL

 PCR MasterMix 12.5μL

 Add sterilized double distilled water to 25 L.

 Pre-denaturation at 94 °C for 5 min, single cycle 94 ° C, 45 s, 50 ° C, 45 s, 72 ° C, 30 s, a total of 30 cycles, 72 ° C extension lOmin end. 5 μL of the PCR product was detected by electrophoresis on a 1% agarose gel.

 3) Purification and recycling

 After the end of the electrophoresis detection, the amplified fragment was purified by agarose gel recovery kit. The purification steps are:

a. After electrophoresis, cut the gel fragment containing DNA. Try to remove excess gel.

b. Cut the rubber block and weigh 3 times the buffer into a 1.5 mL colorless centrifuge tube. c, incubate for 10 min at 50 °C (the direct gel is completely dissolved).

d. After the gel is completely dissolved, check if the color of the solution is yellow. If it is the next step, if not, add IOPL 3 M sodium acetate and mix, the color will turn yellow.

e. Add 1 volume of isopropanol and mix.

f. Transfer the solution to a DNA separation tube (provided by the kit) and centrifuge at 12000 xg for 3 min.

g. Add 0.75 mL of PE buffer to the separation tube and centrifuge at 12000 xg for 3 min.

h. Transfer the column in the separation tube to a 1.5 mL clean centrifuge tube and centrifuge at 17900 xg for 1 min. i. Place the column in a 1.5 mL clean centrifuge tube and add double distilled deionized water to the amount, 17900X _g The purified DNA of interest was obtained by centrifugation for 1 min.

4) Purpose DNA labeling

 Add the PCR fragment recovered from lyg to the PCR tube, add sterile double distilled water to 16 μL; denature in boiling water for 10 min, then quickly put it into the ice bath; mix and add 4 L DIG-High Prime to the denatured DNA. Mix and gently centrifuge, incubate at 37 ° C for 16-20 h (overnight); finally heat at 65 ° C for 10 min, stop the reaction. The control group was supplemented with 15 L of double distilled water using l L control DNA, and the other steps were the same as the labeled group. Dot blot hybridization was used to detect labeling efficiency after labeling was completed. The labeled sample is stored at 20 ° C.

5. Effects of transfection by injection on the fecundity of male and female rats

Six-week-old healthy male rats were divided into two groups, the first group was unilateral testicular injection, and the second group was bilateral testicular injection. First, the mice were weighed and intraperitoneally injected with pentobarbital anesthetic at a dose of O. OOOlg per gram of body weight. While waiting for anesthesia, incubate 2 μί with 4 PL liposome for 20 min. Before use, add 0.5% trypan blue 3 μί. After anesthesia for about 20 minutes, the male rats entered a coma and the mouse abdominal cavity was fixed upward in the surgical disk. Spray the surgical site with 75% alcohol, then wipe it off with absorbent paper, then cut off the hair from the scrotum. The surgeon squeezed the testes into the scrotum with the thumb and forefinger and fixed them. The plasmid complex was injected into the testis tissue and the epididymis with microliter syringes. The unilateral injection group was injected with 9 μL of the complex, and the bilateral injection group was used. 5 μ L。 Side injection 4. 5 μ L. The needle of the micro-injector used in the operation is very sharp. In order to reduce the effect of surgical trauma on the reproductive ability of the mouse, it is not necessary to cut the scrotum for operation. The surgical procedure is shown in Figure 1. At 24 hours after surgery, the two groups of experimental male rats were mated with the normal female rats that were superoved, and the vaginal suppository was examined overnight. The vaginal suppository indicated successful pregnancy. The female rats should pull the ovaries out of the body for surgery. It is very easy to injure the ovaries during the operation and affect the reproductive ability of the female rats. In this experiment, we compare the fecundity of transgenic mothers and normal females, observe the effects of surgery on the reproductive performance of the mothers, and the time required for recovery, in an effort to find a reasonable time for the mother to rest after surgery. 5。 The method of referring to 2.5. After single-cage feeding for 24 hours, the mother rats and the control group were intraperitoneally injected with PMSG (5 IU/only) at 16:00 pm and HCG (5 IU/only) 48 hours later. Immediately after the injection, the mother was caged in the ratio of 1:1 with the normal male rats. The embolization was checked at 7: 00 the next morning, and the mating mother was counted and then single-cage. Let the female rats without embolization continue to mate with the male rats in the cage and check the bolts every morning. After 7 days, the non-pregnant mothers were counted and eliminated. The obtained two sets of data were subjected to a t test to analyze whether the difference in the litter ability between the mother and the control group was significant after the operation. 6. Study on the time of exogenous DNA metabolism after testicular injection of male and female ovary

 After the male rats were injected on both sides for 24h, 48h, 72h, 96h, 120h and 35d, 40d, the cervical vertebrae were dislocated and killed. Each epididymis took about 2 μL of semen and centrifuged 3 times at 2000r/min, using DNase I at 37. After 30 min at ° C, the sperm genomic DNA was extracted according to the method described in the tissue genomic DNA extraction kit. Sperm genomic DNA 1 μL was taken to prepare a PCR reaction system with a total volume of 25 μL. The upstream primer was 5, -TGC TTC AGC CGC TAC CC-3, (SEQ ID NO: 1), and the downstream primer was 3, -AGT TCA CCT TGA TGC CGT TC-5, (SEQ ID NO: 2). The theoretical amplified fragment is 298 kb in length. The PCR primers were synthesized by Beijing Aoke Biotechnology Co., Ltd.

 A 6-week-old sexually mature female rat was intraperitoneally injected with pentobarbital anesthesia at a dose of O. OOOlg per gram of body weight while preparing a plasmid complex while waiting for anesthesia. After the mother had a convulsing anesthetic reaction, the mother was placed on the prone. After entering the coma, the mother's back was symmetrical about the back line about 5 cm below the head, and the surgical site was exposed. Disinfect the skin, open the cortex and muscle layer respectively, pull out the white fat pad with ophthalmology, expose the ovary, and use a micro-injector to inject the plasmid complex 3.5 L into one side of the ovary, if the blue is full The entire ovary, and there is no obvious overflow, indicating that the injection was successful. After the injection, a proper amount of the streptomycin mixture was sprinkled on the wound, the wound was sutured, and the streptomycin was sprinkled again on the epidermis. The procedure is shown in Fig. 2. The ovarian tissue and follicular fluid of the female rats were taken at 24h, 48h, 60h, 72h, 84h, 96h, 120h and lOd, and then the genomic DNA of ovarian tissue and follicular fluid was extracted according to the method of tissue genomic DNA extraction kit. A unique fragment in the plasmid was amplified to preliminarily predict the rate of plasmid metabolism in the body. A total of 25 μL of PCR reaction system was prepared by taking 1 μL of ovarian and follicular fluid genomic DNA, respectively. The upstream primer was 5, -TGC TTC AGC CGC TAC CC-3, (SEQ ID NO: 1), and the downstream primer was 3 '-AGT TCA CCT TGA TGC CGT TC-5, (SEQ ID NO: 2). The theoretical amplified fragment was 298 kb in length, and the PCR primers were synthesized by Beijing Aoke Biotechnology Co., Ltd.

 7. Detection of eGFP expression in embryos of female rats after ovarian injection

 After the ovarian dot is injected with the plasmid and liposome complex, the complex can pass through the cell membrane in a short period of time and enter the ovarian tissue and follicles. In order to detect whether the eGFP gene can be integrated and expressed in mouse embryos through oocytes, the Kunming white mice after surgery were subjected to superovulation, caged and mated with normal male rats of the same line, and the vagina was examined the next day. The test rats were sacrificed 48 h after the cage was placed, and the fertilized eggs were observed under a fluorescence microscope.

Six-week-old sexually mature Kunming white female rats were taken as a group of four. Intravaginal injection of plasmids and Liposomal complex. For the surgical method, see 2.5. At 20h after surgery, the superovulation was performed at 6:00 pm. Immediately after the injection of hCG, the sexually mature males of the same line were mated in a ratio of 2:1. Check the vaginal plug at 7: 00 the next morning. If there is a suppository, it means that the mating is successful and it is placed in a separate cage. The mother rats with the plug were sacrificed by cervical dislocation after 40 h of hCG injection. Place the mouse belly up, place it on absorbent paper, spray 70% alcohol on the abdomen, pinch the abdominal skin of the mouse with tweezers, cut the skin, stretch the head and tail to open the abdominal cavity completely, use ophthalmology tweezers Open the abdominal wall with sharp scissors, find the uterus and ovaries, grasp the upper part of the uterine horn with tweezers, and gently pull the uterus, fallopian tubes, ovaries and fat pads out of the body cavity. This reveals a very thin membrane (the uterine mesentery) that connects the genital tract to the wall of the body cavity with distinct blood vessels. Use scissors to open a mouth on the membrane near the fallopian tube. Use a forceps to stretch the fallopian tubes, ovaries, and fat pads, then use scissors to cut between the fallopian tubes and the ovaries, then cut the uterus near the fallopian tubes, leaving at least 1 cm in length. The oviduct and uterus were removed by sterilized ophthalmology and placed in an embryo observation glass dish (pre-added room temperature M ₂ medium).

 Locate the funnel of the fallopian tube under the microscope, slowly place the end of the fallopian tube with a fine forceps and wash the needle. Gently press the irrigation needle against the bottom of the dish, then rinse the fallopian tube with approximately 0.1 mL of M2 medium. Embryos were harvested using a pipette and the impurities were washed away in fresh M2 medium and placed in a drop of fresh M2 medium. The number of embryos emitting green fluorescence was counted under a fluorescence microscope.

 8. Screening and identification of F1 transgenic positive mice

 The purpose of the transgenic operation is to obtain healthy offspring carrying the gene of interest. After integration of the plasmid and the liposome complex into the oocyte and sperm, whether or not the genome is mutated during gene recombination, so that healthy offspring cannot be obtained, and it is necessary to carry out passage experiments of transgenic mice to confirm.

1) After the postoperative mating arrangement, the male rats were injected into the testes, and the operation was referred to 2.2.6. The female rats were injected with ovarian injection. The male rats were divided into an operation group and a control group, and the mother rats were also divided into a surgery group and a control group. The experimental design was as follows: The rats in the operation group were superoved at the same time as the control group at 12 hours after operation. After the injection of hCG, the normal males of the same sex matured mice in the operation group and the control group were mated in a ratio of 2:1, and the other groups of the surgical group and the control group were also 1:1. The proportion of the cages mated. The embolism was checked at 7:00 the next morning, and the mating mother was counted and then kept in a single cage, so that the mother without embolization continued to mate with the male in the cage. The morning check was performed every morning until 4 days, and the non-pregnant mothers were counted and eliminated. The pups were born for 3 days, placed in a living fluorescence imager for observation, photographed, and the mice were cut toe.

 2) Extraction of genomic DNA After 10 days of birth, the offspring were cut about 1 cm in length and used for genomic DNA and RNA extraction. The genomic DNA extraction method is as follows according to the instructions of the genomic DNA extraction kit:

 The tail was placed in a pre-cooled centrifuge tube, and the tissue pieces were cut as much as possible on ice, and shaken by adding 200 μL of LGA until completely suspended. Add 20 L of Proteinase K and mix. Place in a 56 ° C water bath overnight until the tissue dissolves. Add 200 L GB, immediately mix and invert thoroughly, place at 70 ° C for 10 min, the solution becomes clear, and briefly centrifuge to remove the water drops on the inner wall of the cap. (If the solution is not clear, it means that the cell lysis is not complete, which may result in less or less pure DNA.) Add 200 absolute ethanol, mix well and shake for 15s. After flocculation, briefly centrifuge to remove the tube wall. Water drops.

 The solution obtained in the previous step and the flocculent precipitate were added to the adsorption column and centrifuged at 12000 r/min for 30 s. Pour off the waste and place the column into the collection tube. Add 500 L GD to the adsorption column and centrifuge at 12000 r/min for 30 s. Pour off the waste and place the adsorption column in the collection tube. 700 μL of PW was added to the adsorption column and centrifuged at 12000 r/min for 30 s. Pour off the waste and place the adsorption column in the collection tube. Add 500 rinsing solution to the adsorption column and centrifuge at 12000r/min for 30s. Pour off the waste and place the adsorption column in the collection tube. Place the adsorption column in a collection tube, centrifuge at 12000r/min for 2min, and drain the waste solution. Place the column for a few minutes to thoroughly dry the remaining rinse in the material. Transfer the adsorption column to a clean centrifuge tube and add 50~200 μL of elution buffer to the middle of the adsorption membrane. Place at room temperature for 2~5min, centrifuge at 12000r/min for 2min, collect the solution into the centrifuge tube and set aside.

3) PCR screening of transgenic positive individuals According to the pIRES-eGFP plasmid sequence, in order to exclude endogenous interference, PCR specificity is provided to detect positive individuals for gene integration. Three pairs of primers were designed using the software Primer premier 5, the three pairs of primers were P1 5, -TGC AGT GCT TCA GCC GCT AC-3, (SEQ ID N0: 3), P2 3, -TGT GAT CGC GCT TCT CGT TG_5, (SEQ ID NO: 4); The pair of primers theoretically amplified the eGFP fragment to a length of 454 kb. P3 5, -GAG CTC GGA TCG ATA TCT GC-3, (SEQ ID NO: 5); P4 5, -GCC CGG TCT TCT TGA CGA GC-3, (SEQ ID NO: 6); P5 5' - TGC CTG CTA TTG TCT TCC CA-3, (SEQ ID NO: 7). The P3P5 pair of primer amplified fragments contained the entire reading frame from the CMV promoter to PloyA, and the amplified fragment size was 1953 bp. P4P5 primer pair amplified fragment contains From the IRES to the PloyA reading frame, the entire amplified fragment size was 1429 bp. The PCR primers were synthesized by Beijing Aoke Biotechnology Co., Ltd.

 The PCR reaction was carried out, and the reaction system was as follows:

 Template DNA 1 μL

 10 pmol/L PI 1 μL

 10 pmol/L P2 l u L

 PCR MasterMix 12. 5 μL

 Add sterilized double distilled water to 25 L. Reaction conditions:

 P1P2: Add the mixture by centrifugation according to the above system and put it into the PCR machine. Pre-denaturation at 94 °C for 5 min, single cycle program was 94 ° C, 45 s, 50 ° C, 45 s, 72 ° C, 30 s, a total of 30 cycles, 72 ° C extension lOmin end. The PCR product 5 L was detected by 1% agarose gel electrophoresis.

 P3P5: Add the mixture by centrifugation according to the above system and put it into the PCR machine. Pre-denaturation at 94 °C for 5 min, single cycle program is 94 ° C, lmin, 52 ° C, lmin, 72 ° C, 2 min, a total of 30 cycles, 72 ° C extension lOmin end. The PCR product 5 L was detected by 1% agarose gel electrophoresis.

 P4P5: Add the mixture by centrifugation according to the above system and put it into the PCR machine. Pre-denaturation at 94 °C for 5 min, single cycle program is 94 ° C, lmin, 51 ° C, lmin, 72 ° C, 2 min, a total of 30 cycles, 72 ° C extension lOmin end. The PCR product 5 L was detected by 1% agarose gel electrophoresis.

 4) Southern blot hybridization of PCR products

After the PCR product was electrophoresed, the angle was marked, and 0.25 M HCl was immersed for 18 min. The solution was poured off, washed several times with water, and the solution was poured off. The same operation was carried out for 30 min, and the solution was neutralized twice for 15 min each time. Use a glass plate to build a transfer film platform, and then put 20XSSC saturated filter paper, gel, nylon film with 2XSSC infiltrated 3mmol/L filter paper, then put a stack of dry absorbent paper on the filter paper, set a glass plate at On the absorbent paper, a weight of about 500 g was placed thereon and transferred overnight. After the transfer film was completed, the film was washed in 2X SSC for 20 s, air-dried on a filter paper, and pre-hybridized after baking at 120 ° C for 30 min. Discard the pre-hybrid solution, add the prepared probe solution to a plastic bag, and mix at a hybridization temperature (37 ° C - 42 ° C) for 4 h. After the hybridization, remove the membrane. At room temperature, use the washing solution A. Rinse the membrane twice, 5 min each time, and slowly oscillate during the washing process. Wash the membrane twice with 65 °C washing solution B for 15 min each time, wash the membrane at room temperature for 2 min, pour off the washing solution, and add 100 mL Blocking. The solution was shaken at room temperature for 30 min and the solution was poured off. Add 20mL of equilibration solution, leave it at room temperature for 3min, pour off the solution, add 10mL of color developer, and display it in the dark box. Color, the target strip is colored and then stopped with 50mL TE.

5) Reverse transcription-PCR (RT-PCR) was used to detect the transcription of the transferred gene. Three PCR-positive male and female males were selected, and the testis tissue of the liver, kidney, heart, muscle, tail tip and male mouse and the ovary of the female mouse were removed. Tissue, extract tissue RNA. For specific operation, refer to the instruction of total RNA extraction kit (RNAsimple Total RNA Kit): 1 each sample is about 0. lg, the tissue is ground in liquid nitrogen; 2 add lmL lysate RZ, homogenize with a paddle The homogenate sample was allowed to stand at room temperature for 5 min, and centrifuged at 12000 r/min for 5 min at 4 °C; 3, the supernatant was transferred to a new Rnase-free centrifuge tube, 0.2 mL of chloroform was added, and the lid was shaken vigorously. 15s, stand at room temperature for 3min, centrifuge at 12000r/min for 10 min at 4 °C; 4 After the phase separation of the solution in the tube, take the water phase part and transfer it into a new tube. 5毫升去。 0. 5mL go to the CR tube to add 0. 5mL go to the CR tube to the CR tube to remove the waste solution Protein RD, centrifuged at 12000r/min for 4s at 4°C for 30s, and drained the waste liquid; 6 Add 0.7mL rinse solution RW to the CR tube, let stand at room temperature for 2min, centrifuge at 12000r/min for 4s at 4°C, and discard the waste. 7; Add 0. 5mL rinse RW to the CR tube, let stand for 2min at room temperature, centrifuge at 12000r/min for 30s at 4°C, and pour off the waste liquid; 8 Put the adsorption column back into the 2mL collection tube, 4°C Centrifuge at 12000r/min for 2min, pour off the waste liquid, leave it at room temperature for a while, and evaporate the ethanol. 9 Put the adsorption column into a new centrifuge tube, add 3(^lRNase-free ddH20, place at room temperature for 2min, at 4°C Centrifuge at 12000r/min for 2min. Add 30μί RNase-free dd 0 to the adsorption column, let stand for 2min at room temperature, centrifuge at 22000r/min for 2min at 4°C. After electrophoresis, store at -70°C. The primers were heated in an RNase-free Ep tube at 70 ° C for 5 min, ice bath for 5 min, and added to the following after centrifugation. Substance:

 M-MLV5 X React ion Buffer 5. ΟμΙ

 dNTP (25mmol/L) 1. ΟμΙ

 Rnas in Ribonucl ease Inhibitor 0. 6^L

 M-MLVRT ΐμί

 Nuclease— Free water 14. vL

 The reaction system has a total of 25 L. Heat in a 42 ° C water bath for 1 h, inactivate at 95 ° C for 5 min, ice bath. Used as a template for PCR reactions.

 The PCR reaction system is as follows:

Template DNA 3 μ L 10 pmol/L PI 1 μL

 10 pmol/L P2 1 μL

 PCR MasterMix 12. 5 μL

 Add sterilized double distilled water to 25 L. Reaction conditions:

 P1P2: Add the mixture by centrifugation according to the above system and put it into the PCR machine. Pre-denaturation at 94 °C for 5 min, single cycle program is 94 ° C, 45 s, 52 ° C, 45 s, 72 ° C, 30 s, a total of 30 cycles, 72 ° C extension lOmin end, 4 ° C preservation. 5 μL of the PCR product was detected by electrophoresis on a 1% agarose gel.

III. Results and analysis

The cleavage of the plasmid and the electrophoresis of the PCR product. The plasmid pIRES-eGFP was digested with BamH I and Xho I to obtain two fragments of 1060 bp and 4100 bp. The total length of the plasmid was 5. 2 kb. The plasmid was pIRES-eGFP. Using the design primers, a 298 bp fragment of eGFP in the plasmid was amplified. As shown in Fig. 4, in Maker, there was a distinct bright band at 300 bp, which proved that the fragment had a good amplification.

2. The effect of the injection method on the fecundity of male and female rats. The direct injection of the testis on both testes will affect the reproductive ability of male rats. If unilateral injection is used, the transfection efficiency is theoretically reduced. In order to verify the effect of bilateral injection on the fecundity of male rats, the experimental male rats were divided into two groups: unilateral and bilateral testicular injections. The experimental results are shown in Table 1. The experimental results show that the bilateral testicular testis and the unilateral injection of the testes of the male rats have no significant difference in the ability of the female to conceive. It can be considered that the two surgical procedures have the same effect on the reproductive ability of the male rats, in order to produce more A large number of transgenic spermatozoa increased the number of transgenic positive mice, so the subsequent experiments used bilateral testicular injection. It can also be seen from the experimental results that some male rats are mated with the mother after the operation, and the female rats cannot be conceived after 5 days. In order to confirm whether the spot injection can affect the reproductive ability of male animals for a long time, the male rats with unsuccessful mating continue to observe the study. After 15 days, all the male rats can conceive the female rats. Table 1 List of maternal conceptions

 Subject to

1 ^# 2"3"4" 5 ^# 6 ^ff T 8 ^s 9 ^# 10 ^#孕

Rate % single side note + + + + ― + + — + 35% Shot double side note

 ― + ― + + ― + + + ― + 35% shot

 "+" means vaginal suppository, "1" means no vaginal suppository

 After 7 days, the results of the mother's embolization were as follows: The pregnancy rate of the mother after surgery was 75%, and the pregnancy rate of the normal mother was 86.7%. The litter size is shown in Table 2. It can be seen that surgery has an impact on the fertility of the mother. Table 2 The number of litters in the experimental mother and the control group

 Surgical mother rat normal mother number

 Number of litters

1-1 4 2-1 2

 1-2 9 2-2 7

 1-3 9 2-3 8

 1-4 7 2-4 6

 1-5 5 2-5 5

 1-6 6 2-6 10

 1-7 12 2-7 9

 1-8 4 2-8 6

 1-9 3 2-9 7

 1-10 8 2-10 9

 1-11 6 2-11 3

 1-12 5 2-12 6

 1-13 2 2-13 4

 1-14 6 2-14 9

 1-15 12 2-15 11

 Total 94 Total 102

 Average litter size 6. 2 Average litter size 6. 8

3. Metabolism time of foreign DNA in testis and ovarian tissue At 12 hours after the operation, the male rats were found to be active and normal, with no symptoms of scratching the scrotum, anorexia, and eating foreign bodies, indicating that the surgical procedure had less damage to the rats and the body recovered faster. After the male rats were injected for 24h, 48h, 72h, 96h, 120h, and 35d, 40d, 8 male rats were sacrificed each time to extract testis genomic DNA. The experiment found that the number of male mice with positive test DNA for testicular tissue PCR decreased with time, and the presence of foreign genes could not be detected after 5 days. At the same time, 8 male mice were used for positive detection of exogenous DNA in semen PCR, and the number of male mice decreased with time. It can be seen from Fig. 5 that after the injection, the number of positive mice is slowly decreased. At 35 days after the injection, the number of male mice detected in the semen has a rise, and then the positive rate shows a downward trend, 40d. After falling to a very low level. It indicates that the metabolism time of foreign genes in animal tissue cells is about 5 days, and the differentiation of male germ cells is a coherent process. Therefore, in the process of transfecting germ cells, male germ cells are transfected at various stages, so Positive sperm can still appear after 35 days. These sperms are differentiated from post-transfected spermatogonial stem cells, so in about 40 days, the male mice can produce positive sperm. When the male mouse is operated, the testicular tissue and the epididymis are injected. The purpose of the injection of the epididymis is that the target gene can be rapidly encapsulated with the sperm in the epididymis under the encapsulation of the liposome, so that the male mouse can produce the transgenic sperm in a short time after the operation. The purpose of injecting testicular tissue is to use the complex to transfect the epithelial cells of the seminiferous tubules in the testis, and then transfect the spermatogonial stem cells in the seminiferous tubules. If this process is achieved, then after 5 weeks, the spermatogonial stem cells will develop into sperm. According to the above theory, people can get a large number of transgenic sperm by this method. Through the test results, we can see that the phenomenon of high sperm positive rate occurred 35 days after surgery, indicating that the foreign gene was transfected into spermatogonial stem cells after testicular injection in male rats, and the positive rate decreased significantly after 40 days. Therefore, during the period of 35 to 40 days after surgery, it is also suitable for male mice to mate and produce transgenic animals. The survival rate of the mother after surgery was 92.7%, due to individual differences in the tolerance of the anesthetic. The behavior of the mother rats was observed 12 h after surgery, and the feeding was normal, inactive, and mentally ill. Some female rats scratched the wound, biting the suture, and restless. Three female ovaries were taken, washed with saline, and the follicular fluid was collected. Tissue DNA was extracted from follicular fluid and ovarian tissue, respectively, and then PCR was performed to determine the plasmid metabolism time. The detection situation is shown in Figure 6. At 7 days after surgery, the number of positive individuals detected by PCR in mouse ovarian tissue and follicular fluid decreased. This result makes a reasonable judgment for the mating time of the maternal rats. On the 7th day after the operation, the mother rats should not be used if they cannot be mated successfully with the male rats. In this experiment, the mothers were superoved at 12 hours after surgery. 4. The eGFP expression of oocytes after oocyte spot injection and the eGFP expression of embryos were removed from the cervical vertebrae. The oocytes were collected and placed under a fluorescence microscope at 488 nm. result. After the ovarian spotting injection was performed on the mice in the experimental group, superovulation was performed, and then mating with normal male rats of the same line. The pregnant female rats were sacrificed by cervical dislocation, and the fertilized eggs were collected and placed under a fluorescence microscope. The excitation light was observed at a wavelength of 488 nm and photographed. The results showed that the fertilized eggs obtained from the mice in the experimental group treated with ovate injection could express green fluorescence.

5. F1 generation mouse genomic PCR product analysis Using the primer P1P2, the F1 generation mouse tail tip tissue genome was PCR amplified. As shown in Fig. 8, there was a distinct bright band at 300 bp, which was the same size as the positive control. The band was not amplified in the tail of the control mice. Prove that the target fragment was well amplified. This indicator can be used to initially screen positive mice. The offspring of the test-injected male and normal females are referred to as group A, and the offspring of the ovarian-injected mother and the normal male are referred to as group B, and the test-injected male rats are ovarian-injected. The progeny of maternal mating are called group C. The positive rate of PCR in the tail tip tissue of group A was 40.51% (32/79). The positive rate of PCR in the F1 mice of the B group was 48.94% (46/94). The positive rate of PCR detection in group C was 81.36% (48/59). The positive Blot rates of the three groups A, B, and C were 32.91%, 45.74%, and 71.18% (42/59), respectively. The Fl generation of the A, B and C groups were tested by PCR. The positive rate of transgenic in group C was significantly higher than that in A and B groups. See Table 3 for details.

 Table 3 Comparison of PCR positive rates of F1 generation obtained by three methods

 Sample A 10 0 79 32 26 40.51 32.91 Sample B 0 15 94 46 43 48.94 45.74 Sample c 10 10 59 48 42 81.36 71.18

The primers P1P2 were used to detect the genome of group C F1 mice, and the positive samples were detected. The samples were used for Southern hybridization analysis. The genomes of the positive mice were subjected to PCR using 3 pairs of primers, respectively, and the results were used for agarose gel electrophoresis. The gel after electrophoresis was denatured, neutralized, and then transferred, and the membrane was further hybridized, eluted, and developed. The results are shown in Figure 7, with lane 1 being the negative control and lane 4 being the positive control. Lanes 2 and 3 are samples and are the same size as the positive control. In the hybridization map, the hybrid fragments shown are all a single band.

After RT-PCR, the results are shown in Figure 9, 1 being the negative control, 2, 6 being the heart and liver, respectively, in which no R A transcription was detected. RNA transcription of foreign genes was detected in the kidney, liver, testis, and ovary and tail tip tissues, respectively. It can be seen that the transcription of foreign genes in the F1 viscera is different. Three P1P2 PCR amplification products were randomly selected, purified and recovered, and sent to Beijing Aoke Biotechnology Co., Ltd. for sequencing. The sequencing results were compared with the eGFP gene sequence and found to be completely consistent. The eGFP foreign gene was integrated into the mouse genome.

6. Observation of F1 generation positive mice in living imager

 F1 mice were observed using a living imager from the Experimental Animal Center of Peking Union Medical College Hospital of Beijing Academy of Medical Sciences. The newborn pups were observed by ultraviolet light under the excitation of a stereoscopic microscope, and the limbs and tails fluoresced. In a living fluorescence imaging system, local fluorescence occurs. It can be stated that green fluorescent protein is expressed in young rats. The limbs and tail tips of the mice were observed under a fluorescent microscope, and it was found that these two sites had significant fluorescence.

Discussion and conclusion

 1. The fate of foreign genes entering the organization

With the help of liposomes, the plasmid injected directly into the tissue can smoothly pass through the cell membrane and enter the interior of the cell. However, most of the plasmids that enter the cell may be excreted as foreign bodies as the cells are metabolized. Studies have shown that a large number of plasmids enter the oocyte and are absorbed by ovarian cells, including interstitial cells, granulosa cells, and oocytes at all levels. The foreign gene may be degraded by the nuclease after entering the cell, or it may be killed by the self-protection function of the cell. However, a large number of experimental results show that it is feasible to use this method to produce transgenic animals. In theory, as long as a copy of the plasmid can enter the nucleus and integrate with the chromosome, with the combination of spermatozoa, the progeny carrying the foreign gene will eventually be produced. Therefore, increasing the copy number of the foreign gene as much as possible, and increasing the possibility of entering the germ cell nucleus, while transfecting the male and female germ cells will turn from the idea to a one that improves the efficiency of the transgene. Line method.

 2. Transfection of male germ cells

 Testicular injections can be used to transfect male germ cells at various stages. The mechanism of testicular injection of male germ cells includes two aspects: On the one hand, in the case of dot injection, the needle tip of the injector cuts the seminiferous tubule, and the plasmid/liposome complex enters the fine sperm from the wound. Tube, thereby completing the transfection of spermatogonial stem cells and spermatocytes; on the other hand, the plasmid/liposome complex is injected into the tissue fluid of the testis by injection, and reaches the various tissues in the testis and the epididymis through the body fluid circulation, thereby completing Transfection of germ cells, when the complex is injected into the testis, causes an increase in intratesticular pressure, and this osmosis is more pronounced, which promotes the transfection of the plasmid after the encapsulation of the plasmid by the above two mechanisms. Compared with in vitro incubation, the introduction of exogenous DNA is not inhibited by the seminal plasma during the transfection process, which greatly improves the efficiency of DNA introduction. In vitro fertilization of spermatozoa During the fertilization, most of the sperm head membrane still adheres to exogenous DNA, which affects the vitality of the sperm and leads to the adverse consequences of decreased fertilization ability. The transfected sperm cells are further matured in the epididymis, a process that adjusts the physiological state of the sperm so that its viability is not affected. Since the sperm fertilization ability of the transfected sperm in vivo is not reduced, and the transfected sperm is more than the number of transfected sperm obtained by the in vitro incubation method, the probability of obtaining the transgenic animal is correspondingly increased.

 The occurrence of sperm is a delicate process of continuous synchronization and spatial order differentiation. Since the time from differentiation of stem cells to sperm is approximately constant (about 5 weeks in mice), the cross section of any seminiferous tubule has a set of The typical cell combination of the basement membrane into the lumen, which reflects the continuous wave of waves along the seminiferous spermatozoa. The occurrence of sperm depends on a true stem cell population that has the ability to self-renew and produce the differentiation of the offspring into sperm. Therefore, transfection of spermatogonial stem cells will produce a large amount of transgenic sperm for a long time. In the experiment of the present invention, the male sperm of 35d to 40d after the spot injection is subjected to PCR detection, and a positive result can be obtained: if the foreign gene entering the somatic cell is not integrated, it will be detected after 5 days. Less than, it may be caused by nuclease degradation in cells, or it may be exocytosis of cells and some unknown factors are discharged. Therefore, injections into the testes of male rats were transfected with spermatogonial stem cells, spermatocytes and sperm of male rats.

3. Conclusions After the ovarian injection of the liposome-encapsulated plasmid pIRES-eGFP, the oocytes were harvested and green fluorescence was observed under a fluorescence microscope. At the same time, normal male rats were mated with postoperative mother rats, and 2-cell stage embryos were collected and green fluorescence was observed under a fluorescence microscope. This method indicates that the foreign gene eGFP is integrated into the ovary. The tissue enters the genome of the oocyte and is expressed; the oocyte that incorporates the foreign gene carries the gene into the embryo during fertilization. After the liposome was coated with the plasmid pIRES-eGFP, the testis was directly injected, and the mother was mated with the normal mother. The semen in the vagina of the mother was collected and tested by PCR, and the test result was positive. It was confirmed that the testicular injection method is an effective method for transfecting sperm.

 The offspring obtained by mating the testicular male rats with the normal female rats are referred to as the group A, and the offspring obtained by mating the ovarian-injected mothers with the normal male rats are referred to as the group B, and the ovaries of the testes and the female rats of the male rats are both After the operation, the offspring obtained by mating are referred to as group C. The F1 mice obtained from the three experimental groups were all living. The positive rate of PCR in the offspring of the three experimental groups was as follows: The positive rate of tail-tip tissue of group A mice was 40.51% (32/79); the group B F1 mice passed the tip tissue. The positive rate of PCR detection was 48.94% (46/94); the positive rate of DNA detection in tail tip tissue of group C was 81.36% (48/59), and the positive rate of Southern Blot in sputum, β and C groups was 32.91. %, 45.74%, 71.18% (42/59) (Figure 10). The positive rate of transgenic group in group A, B and C was significantly higher than that in group A and B. The results showed that the germ cells of male and female individuals were transfected in vivo and then mated normally. The positive rate of transgenic generation was higher than that of transfected female germ cells or male germ cells.

Example 2 Maternal Dot Injection Method to Obtain Transgenic Offspring Sheep

 According to the above experimental procedure, the present invention successfully transferred the inhibin gene to the testis tissue and ovarian tissue of sheep by spot injection. The offspring of male testicular male sheep mated with normal female sheep are referred to as group A, and the offspring obtained by mating ovarian-injected female sheep with normal male sheep are referred to as group B. The testis of male sheep and the ovaries of female sheep are both After the operation, the offspring obtained by mating are referred to as group C. The PCR positive rate of the offspring of the three experimental groups was as follows: The positive rate of PCR in the tail tip tissue of the F1 sheep was 46. 2% (6/13). The positive rate of PCR detection in the F1 sheep of the B group was 38.5% (5/13), and the positive rate of PCR in the tail tissue of the C group was 66.7% (4/6) (Fig. 11). In the positive rate of the three groups of progeny, the positive rate of transgene after the male parent was significantly increased compared with the surgery group of the female parent and the male parent.

Claims

Rights request

 1. A new method for improving the efficiency of transgenic animals, including the following steps:

 1) preparing a plasmid with a foreign gene;

 2) introducing a plasmid carrying a foreign gene into a testis tissue of a male animal by spot injection;

 3) introducing a plasmid carrying the foreign gene into the ovarian tissue of the female animal by spot injection;

 4) Male testicular male animals are mated with female animals to obtain progeny of transgenic animals.

 2. A method of increasing the efficiency of transgenic animals according to claim 1, wherein said dot injection method directly injects a foreign gene into the testis and ovarian tissue of the animal.

 3. A method of increasing the efficiency of transgenic animals according to claim 1 wherein said animal is a mammal.

 4. A method of increasing the efficiency of transgenic animals according to claim 1, wherein said method can be used to increase the positive rate of foreign genes carried by progeny animals.

 5. A new method for increasing the efficiency of transgenic mice, comprising the following steps:

1) preparing a plasmid with a foreign gene;

 2) introducing a plasmid carrying the foreign gene into the testis of the male mouse by spot injection;

 3) introducing a plasmid carrying the foreign gene into the ovary of the female mouse by spot injection;

 4) Select the male rats after 1-5 days and 35 to 40 days after the operation of the injection injection, and test the female rats 1-7 days after the injection;

 5) The selected male mouse and the female mouse are mated to obtain progeny of the transgenic mouse.