technology that targets genes
Gene pooping is an experimental tool for targeted alteration of genetic information of a species based on embryonic stem cells (ESCs) and homologous recombination. In vitro homologous recombination is used to obtain ESCs with a pre-designed mutation, i.e., mid-target ESCs, and then these genetically modified ESCs are injected into blastocysts to obtain chimeras, which are then mated with wild-type individuals and, if the ESCs have been integrated into the germ line, the resulting offspring will have a chimera carrying a mutated chromosome.
If the E S cells are integrated into the germ line, a heterozygous individual carrying one mutant chromosome will be obtained in the offspring generation, and finally a pure individual carrying two mutant chromosomes will be obtained in the offspring generation through genetic breeding. Since T h o m a s and Capecchi accomplished the world's first gene targeting experiment in 1987, gene harrowing has become a widely used routine technique.
Author: Xuedao Pei, this experiment is from "Stem cell experiment guide".
Operation method
technology that targets genes Move PCR method to obtain homologous arm sequences The first step in constructing a raking vector is to obtain the homology arm sequence for homologous recombination. The homology arm sequence can be obtained by screening the mouse genome library, and now the mouse genome sequencing has been basically completed, so it can be obtained by the simpler and more effective polymerase chain reaction (PCR) method. In order to improve the efficiency of homologous recombination, the homology arm sequence should preferably come from the ES cell line to be manipulated (Rigle et al., 1992), and the length of the sequence should generally be 4--10kb (Deng et al., 1992), so high-fidelity DNA polymerase, which can amplify longer fragments, is used for PCR. Therefore, high-fidelity DNA polymerase, which can amplify longer fragments, is used for PCR. The main factors to consider that affect the success of PCR are: (I) D N A template. The integrity and purity of the DNA template is critical, and its average length should be at least three times the expected length of the PCR amplification product, and the OD2602/OD280 ratio should be around 1.8 to allow for the inclusion of the P C R inhibitor. (2) DNA polymerase. A number of companies have developed high-fidelity DNA polymerases suitable for amplifying longer fragments. The author has used the genome of ES cells extracted by method 8.2.5 as a template, and used Stratagene's PfuUltm polymerase to successfully amplify a fragment of 4.2k b by the Touchdown PCR method, and found that there was not a single mutation after sequencing. Nested PCR using Pyrobest DNA polymerase from TaKaRa successfully amplified a 2.6k b fragment, and no mutation was found after sequencing. (3) Primers. General principles of primer design must be followed. The primers used for the longer PCRs are generally slightly longer (25--30 bp) than those for the standard PCRs. The annealing temperatures of the two primers should be approximately equal. A suitable restriction endonuclease site can be introduced at the 5' end of the primer for subsequent DNA manipulation. Please follow the conventional molecular cloning method. The following is the method used by the author to obtain the homology arm sequence, which is for reference only. 8.1.1.1 T o u c h d o w n P C R 8.1.1.2 Purification of P C R Products of the liquid, reposition the column on the collection tube. (8) Add 50ul of sterile water, incubate with Imin at room temperature, centrifuge at 16,000g for 1min, and store the eluate at -2℃. PCR product tailing Connection of PCR products to pGEM-Tesay carrier The pGEM -T Easy vector is a Promega product (Cat.# A 1360). (1) Use the Previbration Suspension 2x Quick Connect buffer. Transformation of linkage products (2) Take 5ul of the linkage product and mix it with the IOP receptor cells on ice, and ice bath for 30 min. (3) Heat-excite at 42℃ for 90s, then put on ice for 2--3 min. (4) Add 800ul L B medium and incubate at 37℃ with 150r/m i n vibration for 45 min. (5) Centrifuge at 7000r/m i n for 2 min, discard the supernatant of 800ul, mix the remaining IOOul with the precipitate, and spread evenly on the L B plate containing suitable resistance. (6) Incubate at 37℃ for 16~18 h, and select the transformants for digestion and sequencing. The joining of long DNA fragments can sometimes be an obstacle to in vitro DNA manipulation. In my experience, the problem is often solved when DNA fragments are recovered using Promega or Qiagen agarose gel recovery kits and ligated using TaKaRa DNA ligation kits (DNA Ligation Kit Ver.2,1; Catalog No. D6022). Cleavage and Recovery 2ug of p GEM ®-TEasy Plasmid containing the insert and the target plasmid should be digested with a suitable restriction endonuclease. Recover the insert and targeting plasmid from the agarose gel as described above. The target fragment is connected to the targeting vector. The DNA connection kit from T a K a R a Company was used. (1) Mix the target fragment with the targeting vector at a molar ratio of 3 : 1, in a total volume of 5 to 10 ul, and add an equal volume of solution L and mix well. (2) React at 16 °C for 30 min and transform. Adding 1/10 volume of Solution III can enhance the transformation efficiency by 2-5 times. (3) Incubate at 37℃ for 16~l8 h, and select the transformants for digestion and sequencing. Plasmid extraction 8 . 1 . . 2 . 4 Target Carrier Linearization Higher homologous recombination efficiency can be achieved by using linearized DNA (Hasty et al. 1992). Targeting vectors should be linearized before transfection of ES cells by electroporation, so when designing targeting vectors, there should be a single cleavage site at the end of one of the homology arms. The HSV-TK gene is usually chosen as the negative selection gene in the vector, and if the TK gene is at the end of the targeting vector, it is often destroyed during transfection. It is therefore desirable to have a single cleavage site in a homologous arm away from the TK gene so that the TK gene is not at the end of the vector after linearization. Please follow the conventional molecular cloning method for digestion and ethanol precipitation, and the following procedure is for reference. (6) Centrifuge at 12 000r/m i n room temperature for 5 min, carefully discard the supernatant in a sterile ultra-clean bench and dry the precipitate. (7) Resuspend DNA with about 500 sterile water and keep at -20°C. Methods for screening and characterizing mid-target E S cells are well established. The following methods have been used successfully by the Developmental and Disease Genetics Research Laboratory, Institute of Bioengineering, Academy of Military Medical Sciences. Preparation of fibroblast trophoblasts from primary mouse embryos Both S-TO cell lines and primary mouse embryonic fibroblasts can be used to support the growth of embryonalcarcinoma (E C) cell lines and ES cell lines. Suemori and Nakatsuji (1987) compared the growth of S-TO and protoplasmic fibroblasts with that of S-TO. (1) Female mice that are 13.5 to 14.5 days pregnant are executed, the abdominal cavity is opened, and the embryos are removed and placed in a sterile 90m m diameter petri dish. (2) Remove the yolk sac, amniotic membrane and placenta, and wash the embryos twice in fresh PBS. (3) Remove the head and viscera of the embryo with a pair of forceps, cut the fetus into small pieces (about 1 m m 3 ) with sterilized scissors, and wash in PBS until the liquid is almost colorless. (4) Transfer the pieces of fetal mouse to a 15 ml centrifuge tube containing PBS and centrifuge for 5 min at lOOOr/m i n . (5) Discard the supernatant, add 5 ml of 0.25% trypsin, leave at 37°C for 30 min, add DMEM medium containing 10% fetal bovine serum, and blow repeatedly with a pipette. (6) To remove large cell clumps, flow the cell suspension through a 2,000-mesh sieve, and then centrifuge for 5 min at lOOOr/m i n . (7) Discard the supernatant, resuspend the cells by adding feeder layer cell culture medium, transfer the cell suspension to 150 m m dishes, and incubate in a cell culture incubator containing 5% C02 at 37 ℃ for 2--3 days, and the embryonic fibroblasts have grown all over the dishes on the third day. The cells are usually full of embryonic fibroblasts by the third day. This is recorded as generation O (P0). The cells can be frozen in medium (15% FCS, 10% DMSO, 75% DMEM). (8) When the fourth generation of cells was full grown, replace the fresh feeder layer cell culture medium, add mitomycin C (mit ○ m y d n C) with a final concentration of 100 ug/ml, and continue to incubate at 37 ℃ for 2--3 h to stop the growth. (9) PBS was washed 5 times, digested with 0.25% trypsin and counted. (10) LOOOr/m i n centrifugation for 5 min, add the appropriate volume of freezing medium, so that the cell density of 2xl 0 6 cells/ml, blow with a pipette and mix well, then freeze and reserve. Usually, a petri dish with a diameter of IO c m needs to be spread with IxlO6 cells to establish a serovar layer. The following formula can be used to prepare 600m l of feeder layer cell culture medium: D M E M 500 ml Glutamine 6 ml Non-Essential Amino Acids Solution (10mol/L, IOOx) 6 ml Penicillin-streptomycin solution (5000U penicillin, 5m g streptomycin/100 ml) 6 ml Mercaptoethanol 4 ul Fetal bovine serum 90 ml Filter with disposable filter bottle and set aside in 4℃ refrigerator. Add 1000U/m l leukemia inhibitory factor (LIF) to this medium, and it can be used for the culture of ES cells. Reagents for ESC culture (1) Water: The quality of water is critical, and distilled water filtered through a Millipore-Q water purification system or purchased ultrapure water is recommended. (2) D M E M : D M E M dry powder medium with high sugar (4.5 g/L ) and L-glutamine (4.0 mmol/L ) was used as the basic medium for the cultivation of E S cells. The dry medium is dissolved in ultrapure water, adjusted to a p H of 7.2 with HCl, and then filtered through a 0.22 um microporous membrane into a sterile container. DMEM is also available in purchased liquid media and can be stored at 4°C for 2 to 4 months. Because glutamine in DMEM is unstable and easily degraded, 2 mmol/L glutamine must be added to DMEM after 14 days of storage. (3) O.lmmol/L non-essential amino acids (lOOxstock). (4) lOOug/ml penicillin-streptomycin. (5) 200 mmol/L L-glutamine (lOOxstock). (6) 15% Premium Fetal Bovine Serum: Fetal bovine serum is an important factor in the growth status of E S cells. Excessive concentration of (7) 10-4mol/L β-mercaptoethanol (IOOxstock): The addition of β-mercaptoethanol to ES cell culture medium promotes the division and proliferation of embryonic cells. Some E S cell lines, such as T C -I E S cells, are dependent on A mercaptoethanol, and E S cells cannot survive without the addition of β-mercaptoethanol. (8) lOOOU/m l Leukemia inhibitory factor (LIF). (9) Ca2+ and M g 2+ free P B S buffer: IL P B S is prepared with the following reagents, IOg NaCl, 0.25 g K C 1, 1.44 g Na2 H P O 4, 0.25 g K H 2P O 4, p H adjusted to 7.2, and autoclaved. Purchased P B S buffer with pH 7.2 may also be used. (10) Trypsin: 0.25% trypsin, 0.02% E D T A . (11) 0.1% gelatin. Routine culture of ES cells (1) Treat the petri dish with 0.1% gelatin and prepare the feeder layer as described above. (2) Discard the original medium and wash once with PBS. (3) Add 0.25% trypsin-EDTA solution, digest for 3-5 m i n , then add DMEM high sugar medium containing 15% fetal bovine serum to neutralize the trypsin. (4) Blow gently with a pipette until it becomes a single-cell suspension. (5) Transfer the cell suspension to a new cell culture dish containing the feeder layer at a ratio of 1 : 3, mix well, and place in a 5 % C 0 2 , 3 7 °C incubator. (6) Change the culture medium once a day, and the cells can be passed on after 3 days. Before changing the solution, preheat the medium or the desired solution to 37°C or room temperature. Passaging should be performed before the peripheral appearance of differentiated endodermal cells in the ESC clone. The most common and successful method of transfecting ES cells is by electroporation. Transfection of ES cells by electroporation will result in the death of up to 50% of the ES cells at the desired transfection efficiency. Factors affecting transfection efficiency and cell viability include voltage, ion concentration, DNA concentration and cell density. All of these factors must be considered and pre-tested to achieve the desired transfection efficiency and cell viability for different ES cell lines. The following is an electroporation method based on TCl E S cells cultured on primary embryonic fibroblasts: (1) Transfection experiments were performed on day 2 (36 h) after passaging of resuscitated E S cells. (2) Change to fresh medium 2 h before transfection. (3) Dispose of the medium and rinse the petri dish twice with PBS. (4) Add 1.5 ml of medium to each 90-mm diameter Petri dish. (4) Add 1.5 ml of 25% trypsin-EDTA solution to each 90-mm diameter Petri dish and let it stand at 37°C for 3~5 min. (5) Add 3.5 ml of ES cell culture medium and aspirate repeatedly with a pipette 20 times. Determine the total number of cells with a blood cell counter plate, usually each raking vector requires about 2x07 cells per transfection. (6) Centrifuge the ES cell suspension at 1000r/m i n for 5 min. Aspirate the supernatant and resuspend the cells in 10m l of PBS. (7) Re-center at 1000r/m i n for 5 min. Resuspend cells in P B S to achieve a cell density of 2xl07 cells/ml. (8) Mix 30--50ug linearized targeting vector DNA with I m l cells, load into electroporation tank and leave for 5m in at room temperature. (9 ) Electroporate in a gene pulser (600V, 25uF) and leave at room temperature for 1min. (10) Mix the cells in the electroporation tank with 7 ml of fresh ES cell culture medium and distribute into 4 90-mm diameter dishes full of trophoblast cells. (11) Spread 40 ul of untransfected cells in a 90 m m dish as a control. (12) After 24 h, ES cell screening medium was changed to ES cell screening medium containing G 418 (280ug/m l) and gancyclovir (2umol/L), and fresh screening medium was changed daily thereafter. Usually, clones can be picked after 7 days of transfection. (1) Cultivate the transfected ES cells in drug screening medium for 7-8 days, then discard the medium, wash with PBS once, and replace with IOmI PBS. (2) Adjust the 2,000,000 automatic pipette to 2,0ul, and select the resistant E S clones under the microscope with the tip of a gun. (3) Transfer each clone to an empty 9 6-well culture plate. (4) Add 50ul of 0.25% Trypsin-Edta solution to each well of a 96-well culture plate and incubate at 37°C for 3~5 min. (5) Add 100ul of ES cell culture medium to each well. Adjust a 100 ul 8-channel automatic pipette to 80ul and blow repeatedly with the tip of the gun to make a single cell suspension of the ES clones. Aliquot the single-cell suspension of each E S clone into two 96-well culture plates pre-inoculated with trophoblast cells. The position and order of the clones are identical in both plates. (6) Replace the ES cell culture medium with fresh one every day and freeze one of the 96-well plates after 2-3 days of incubation. (7) Transfer the ES clones from the remaining 96-well plate to a 24-well plate pre-inoculated with trophoblast cells after trypsin digestion, and extract the genomic DNA for identification. A single test usually involves the screening of 100-1000 ES clones. Freezing ESCs directly in 96-well plates greatly reduces the effort and cost of the experiment. (1) Discard the medium and add 100ul of PBS. (2) Dispose of PBS, add 50ul of 0.125% trypsin-EDTA solution diluted with PBS, and incubate at 37°C for 3--5 min. (3) Add 1,000ul of freezing medium (15% DMSO, 20% fetal bovine serum, 65% DMEM). (4) Disperse and mix the cells with an eight-channel automatic pipette. Seal the 96-well culture plate with sealing film and seal it in a plastic bag, and mark the surface of the petri dish and the plastic bag. (5) In order to slow down the freezing speed, put the petri dish into a foam box, and then put it into a -80°C refrigerator for freezing. If it is determined that PCR screening is to be used, the homology arm length on one side of the rake vector should usually be less than 3kb when designing the rake vector. A short arm with a length of 0.6 to 1.2kb is easiest to detect by PCR. If the homology arm sequence is too long, try using long-distancePCR. To detect the occurrence of a correct homologous recombination event, one primer for the PCR reaction must be designed on the gene, and the other primer should be designed to span the short arm of the target vector, i.e., the endogenous gene fragment on the outside of the target vector. (18) Place the filter membrane in two sheets of filter paper and dry it, wrap the membrane in plastic wrap, and place the membrane in an X-ray film folder in the darkroom at -700°C with an intensifying screen for 12-48 h before developing the image. (1) Remove frozen 9 6-well plates and incubate at 37T to thaw the cell suspension as soon as possible. (2) Transfer the cell suspension into a 24-well plate inoculated with the feeder layer and incubate in an incubator at 37℃ with 5% C02, and change the solution the next day. (3) After the ES cells have grown to a suitable density (usually 3 days), digest the cells and transfer the cell suspension to a 6-well plate for further cultivation. (4) After the ES cells have grown to a suitable density (usually 3 days), digest the cells and transfer the cell suspension to 3 6-well plates for further cultivation. (5) After the ESCs have grown to a suitable density (usually 3 days), freeze the cells in 2 wells and use the remaining well either for blastocyst injection to prepare chimeric mice to study the function of the target genes at the overall level, or expand the culture to conduct a new round of targeting experiments in order to obtain the ESCs carrying the two mutant chromosomes to study the function of the target genes at the cellular level. As for the specific operation of blastocyst microinjection as well as embryo transfer, readers are advised to refer to the relevant chapters of this book or related literature, which will not be repeated here. For more product details, please visit Aladdin Scientific website.
minimize the amount of P C R inhibitors.
(7) Add 500ul of membrane wash solution, centrifuge at 16000g for 5min and place the column on a new 1.5ml centrifuge tube.
Take l--7ul of purified PCR product, add 1ul of IOxTaq DNA polymerase reaction buffer (containing MgCl2), lul2 mmol/L d ATP, lul Taq DNA polymerase, add water to make up 10ul, incubate at 70℃ for 15--30 min. Incubate at 70℃ for 15-30 min.
(1) Prepare receptor cells by conventional calcium chloride method. If the requirement is not met, the receptor cells of Tsinghua Tianwei Times Company can be used.
To prepare plasmids for electroporation transfection, the Qiagen PlasmId Midi Kit (Cat. No. 12143) is preferred. The copy number of the targeting vector is sometimes significantly reduced due to the insertion of long homology arm sequences, and the culture volume should be increased in this case. 
Suemori and Nakatsuji (1987) compared the effects of S-TO and primary mouse embryonic fibroblasts on mouse ESCs and found that primary mouse embryonic fibroblasts were more suitable for mouse ESC culture. Embryonic fibroblasts isolated from various mouse strains did not differ significantly in their ability to support the growth of mouse ESCs. Embryonic fibroblasts used for targeting experiments must be screened for resistance and can usually be isolated from transgenic mice or knockout mice carrying resistance genes.
Cells can be frozen in freezing medium (15% F CS, 10% DMSO, 75% DMEM) at a density of 2xl06 cells/m l, resuscitated when needed, and cultured in 1 : 3 passages.
Too high a concentration of fetal bovine serum is not favorable to the growth of E S cells, and different lots of fetal bovine serum have different effects. Therefore, to avoid lot-to-lot variation, a suitable batch of fetal bovine serum is often selected and frozen in bulk at -20°C for backup. Embryonic stem cell grade fetal bovine serum is available from serum suppliers and has been screened for its ability to maintain proliferation of ES cells without differentiation.
