somatic cell nuclear transplantation (SCNT)
somatic cell nuclear transplantation (SCNT)
Animal cell nuclear transfer/nuclear transplantation refers to the microscopic manipulation of the nucleus of a donor cell into a mature oocyte or early syncytium that has been denucleated to form a new nuclear recombinant, which divides, differentiates, and develops into a new individual in the mother's body. Depending on the source of the donor cells, nuclear transfer can be divided into embryonienuclear transfer, embryonie stem cell nuelear transfer and somatie cell nuclear transfer. ). Both embryonic cell nuclear transfer and somatic cell nuclear transfer can be categorized into primary nuclear transfer and secondary nuclear transfer according to the number of transplants. Multiple generation nuclear transfer, also known as serial nuclear transfer or recloning, refers to the use of nuclear transplanted embryonic cells as the donor cells for nuclear transplantation, and the resulting embryo is called the second generation of nuclear transplanted embryos. In this method, multiple generations of embryos can be obtained, in which the nuclear transfer with embryonic cells or somatic cells is called primary nuclear transfer, and the nuclear transfers in the subsequent generations are called secondary nuclear transfer. For animal cloning, the number of clones produced by embryo splitting is very limited, so the cell nuclear transplantation has become an effective method for producing cloned animals, and people often refer to the cell nuclear transplantation technology as animal cloning technology.
Principle
The principle of nuclear cell transplantation is that all nuclei of cells originating from the same embryo contain exactly the same genetic information as the fertilized egg, and have the same full potential to guide the development of the individual in terms of genetic composition as the fertilized egg.
However, as the embryo develops and the cells differentiate, some of the genomes of the nuclei change, resulting in these genomes not being able to revert to their former state, and thus losing their nuclear totipotency. However, they contain all the genetic information of the organism and, under the regulation of specific environmental factors, can be restored to the fertilized egg-like state and develop from scratch into a complete biological individual.
Appliance
Animal cell nuclear transplantation technology provides a new technical means for livestock breeding, transgenic animal production, protection of endangered animals, study of nucleoplasmic relationship, genomic imprinting research, as well as the exploration of cellular senescence and differentiation mechanisms.
Operation method
somatic cell nuclear transplantation (SCNT)
Principle
The principle of nuclear cell transplantation is that all nuclei of cells originating from the same embryo contain exactly the same genetic information as the fertilized egg, and have the same full potential to guide the development of the individual in terms of genetic composition as the fertilized egg. However, as the embryo develops and the cells differentiate, some of the genomes of the nuclei change, resulting in these genomes not being able to revert to their former state, and thus losing their nuclear totipotency. However, they contain all the genetic information of the organism and, under the regulation of specific environmental factors, can be restored to the fertilized egg-like state and develop from scratch into a complete biological individual.
Materials and Instruments
Equipment: Move The basic process of the micro fertilization technique can be divided into the following steps: 1. Preparation of nuclear donor cells The type of donor cell, as well as the cycle and state in which it is located, can affect the success rate of nuclear transplantation. The types of cells that can be used as donors include: early developmental embryonic cell oocytes, embryonic stem cells, cultured fetal skin fibroblasts, and even somatic cells from various tissue sources in adult animals. 2. Nucleation of recipient oocytes (1) blind suction method: blind suction with a microfine glass tube in the first polar body under the first polar body, suction to remove the first polar body and in the middle of the division of the chromosome and the surrounding part of the cytoplasm. Usually, the time to denucleate oocytes by blind aspiration should be chosen as far as possible when the first polar body is just discharged, and for bovine oocytes in IVM, it is usually 18-20 hours after IVM. (2) Half-egg method: cut the oocyte into two halves, remove the half containing polar bodies, and then use the half without polar bodies for nuclear transplantation. The specific operation method is as follows: transfer the oocyte to a 35 mm Petri dish containing PBSA, and divide the zona pellucida in two steps, i.e., make a small incision on the zona pellucida first, and then enlarge the incision with another cutting needle, thus dividing the zona pellucida. After the zona pellucida was cut, it was transferred to a 35 mm Petri dish containing PBSA and 5 μg/ml cytochalasin B and acted for 3 to 5 min, and then fixed with a fixation needle, the dividing needle entered into the perivitelline gap opening of the zona pellucida and fixed this needle against the zona pellucida, and slowly aspirated the oocyte fluid. When half of the oocyte fluid was aspirated, the dividing needle was slightly rubbed against the cutting edge of the zona pellucida to achieve complete division, and the oocyte fluid in the needle was transferred into the prepared empty zona pellucida and stained with Hoechst33342, and observed under the fluorescence microscope, and those that did not fluoresce were used as receptor oocytes. (3) Centrifugal denucleation: The nucleus of the oocyte without zona pellucida can be thrown to one side by centrifugation and finally detached from the oocyte. (4) Fluorescence-guided denucleation: The oocytes were first stained with Hoechst33342, and then the location of the nucleus was determined under the fluorescence microscope, and then the aspirated cytoplasm or the denucleated oocytes were observed to see whether they contained nuclei or not, in order to ensure the success rate of denucleation. (5) Terminal denucleation method: After activating the oocyte so that it is at the end of the MII stage, the chromatin and a small amount of surrounding cytoplasm underneath it can be removed when the second polar body is discharged. (6) Chemical induced denucleation method: in 1993, Fulka et al. used etoposkle and radiolytic ketone to deal with mouse oocytes in the middle of the first meiotic division, the chromosomes are closely bound to each other after treatment, not easy to be separated, and form a chromosome complex, and the chromosome complexes are discharged along with the first polar body thereafter, which makes the denucleation of the oocyte up to 90% success rate. (7) Cutting and denucleation method: first use plant lectin (PHA) to deal with oocytes, so that the discharged first polar body and oocytes adhere together. Streptavidin was used to digest the oocyte to remove the zona pellucida, and then in the operating solution containing cytochalasin B, the oocyte was divided into two along the parallel direction of the tangent line intersecting the polar body and the oocyte, and this half of the oocyte adhering to the polar body was removed. Nuclear transplantation is accomplished by electrofusion of the two polar body-less halves of the oocyte with a donor cell. (8) Hyperosmotic treatment of nuclear method: 3% sucrose treatment of mouse oocytes, due to changes in the osmotic pressure of the solution, so that the refractive properties of the chromosomes in the region of the change in the location of the nucleus can be clearly observed under the ordinary inverted microscope, and accurately remove the nucleus. (9) Functional denucleation method: Oocytes are stained with DNA-specific Hochest and then irradiated with ultraviolet light to inactivate the DNA breaks, but prolonged ultraviolet light irradiation reduces the viability of oocytes. 3. Nuclear transplantation According to the different sites where the nucleus of the donor cell is moved into, it can be divided into two categories: subband injection and intracytoplasmic injection. (1) subband injection: the nuclear donor is placed in a droplet of operating fluid, and a transplantation needle with a diameter close to the size of the oocyte ball is used to suck up a separated complete oocyte ball or cell, and inject it into the perivitelline space of the egg along the incision left in the zona pellucida of the oocyte when the nucleus is removed, and make the donor cell come into contact with the plasma membrane of the egg. When aspirating the donor cell, the integrity of the cell membrane should be ensured to facilitate the adhesion and subsequent fusion of the donor cell membrane with the oocyte plasma membrane. (2) Intracytoplasmic injection: The nucleus is injected directly into the oocyte plasma with a 5-8 μm diameter needle, usually along the opening left by denucleation. The ambient temperature of the operation is 30-35 °C. The nucleus is injected directly into the oocyte plasma with a 5-8 μm diameter needle. Cell fusion The reconstructed embryo of the oocyte injected under the belt must be fused, so that the nucleus of the donor cell enters into the cytoplasm of the recipient cell to form the reconstructed embryo. According to the different methods of cell fusion, it can be divided into the following types. (1) Virus-mediated fusion: A variety of viruses can mediate cell fusion, among which Sendai virus is more commonly used in animal cell fusion. The inactivated Sendai virus can attach to the cell membrane, causing disruption of the membrane of the contacting part of the cell, forming a channel, and thus cytoplasmic fusion of the two cells. Donor cells and Sendai virus solution with 2500~2600 hemagglutination units (HAU/ml) are injected together under the zona pellucida, and the reconstructed embryo can be fused within 15~30 minutes. This method has an effect on embryo development, is prone to contamination, and is ineffective for some species, so it is less commonly used. (2) Chemical fusion: PEG-mediated cell fusion, the process of which is to depolymerize the cell membranes first, so that the cell membranes at the site of contact will fuse. (3) Electrofusion: It can effectively fuse the plasma membrane of the donor cell with that of the recipient oocyte, and can also activate the oocyte. 5. Activation of reconstituted embryos (1) Chemical activation: It is usually used for the activation of reconstituted embryos that have already undergone fusion by other means, and is therefore used for post-activation. The reagents commonly used for chemical activation are as follows. (1) Ethanol: Put the recombinant eggs to be activated into the culture solution containing 7% ethanol, and treat them at 37 ℃ and CO2 incubator for 5 minutes or at room temperature for 7 minutes, then rinse them and incubate them. (2) Lonomycin (ionomycin): the concentration of 5 μmol/L was used, and the treatment time was 4-5 minutes. This method can rapidly increase the concentration of Ca2 in the cytoplasm. (3) Calcium ion carrier A23187: The concentration of 5 μmol/L was used, and the treatment time was 5 minutes. (4) 6-DMAP (dimethylaminopurine): using a concentration of 1.9 to 2 mmol/L and a treatment time of 3 to 5 hours or longer. 6-DMAP can slowly raise or maintain the Ca concentration in the cytoplasm. (5) Staurosporine: use a concentration of 2 μmol/L and a treatment time of 15 to 30 min. (2) Electroactivation: When activated by transient high-voltage current, it caused the temporary formation of small pores or increased permeability of the cell membrane, so that ions and small molecules inside and outside the cell could be exchanged through these small pores, and as a result, significant transmembrane Ca inward flow was produced, thus activating the oocytes. For more product details, please visit Aladdin Scientific website.
①Fixing needle
② syringe needle
③Capillary glass tube
④ Needle puller
⑤ Needle grinder
⑥Ovipositor
⑦Cell culture box
Reagents:
①Material: animal
②Hyaluronidase
③Sperm incubation solution
④PVP
⑤Paraffin
⑥Dithiothreitol (DTT)