Over the past ten years, research on gene function has yielded fruitful results, which has led to advances in genetic engineering, gene therapy, and monoclonal antibody technology, while revolutionary technological advances in animal reproductive biology have led to sheep, Successful cloning of animals such as cattle, monkeys and mice. The development of electroporation techniques and instruments has played an important role in these achievements, allowing humans not only to clone animals from embryonic cells, but also to obtain cloned animals from fully differentiated mature cells. The new genetic manipulation and reproductive technology will bring important scientific achievements to the 21st century. Electroporation technology is the use of a pulsed electric field to change the state and permeability of the cell membrane to achieve the purpose of introducing DNA into cells and promoting cell fusion. This technology is currently applied on the one hand to gene transfer of bacteria, fungi, plants, insects and mammalian cells, on the other hand to cell fusion for the preparation of hybrid cells and animal clones. 1. Application in gene transfer and hybridomas 1.1 Animal and insect cell transfection Electroporation technology was first used in the early 1980s to introduce DNA into a variety of animal cells [1], compared to traditional calcium phosphate and liposomes. Dyeing and electroporation have many advantages such as simple operation and high transfection efficiency, especially for those cells that are difficult for other methods to have effect, but its influence factors are also more. Electric field strength: When the voltage is too low, the change of the cell membrane is not enough to allow DNA molecules to pass through, and when the voltage is too high, it will cause irreversible damage to the cells. For most mammalian cell cells, a voltage of 250-2500 V/cm can be effectively transfected [2,3]. Electric pulse shape and length: The shape of the electric pulse is mainly exponentially attenuated and square wave. Most of them take 20-100 milliseconds. Buffer: Non-ionic buffers such as mannitol and sucrose are usually used, but HEPES buffers have been reported to have higher transfection efficiency and serum transfection efficiency has also been improved [4]. Other factors such as transfection temperature, DNA concentration and conformation can affect the transfection effect. 1.2 E. coli and yeast transformation Electroporation began in the late 1980s to transform E. coli [5]. Because of the relatively small size of the bacteria, E. coli usually requires 4000 to 20,000 V/cm compared to DNA introduced into animal cells. Pulse intensity can be effectively transfected. The highest transfection efficiency of chemically competent cells can only reach 106-108 transformants per microgram of DNA, whereas electroporation can reach the level of 109-1010 transformants, which is 10-100 times higher than the former. It is critical to work with higher conversion rates such as cDNA libraries. Yeast electrotransformation overcomes the deficiencies of the lithium acetate and protoplast method and the low conversion rate, and the efficiency is significantly improved [6]. 1.3 Cell fusion preparation Hybridoma cell fusion is mainly used to generate hybridoma cells [7, 8]. In the preparation of monoclonal antibodies, hybridoma cells are conventionally produced using the PEG fusion method, which has many limitations in modern production. The electrofusion method can prepare hybridomas in batches and efficiently in a large scale, shortening the entire production cycle. In addition, electrofusion is also widely used in the preparation of other hybrid cells in cell biology studies [9]. 2. Nuclear Transplantation and Activation in Embryonic Engineering Electroporation technology has played a key role in the simple electroporation of embryonic stem cells and the electroactivation of nuclear transfer embryos. The following are some important applications. 2.1 Parthenogenetic, tetraploid, and chimera 2.1.1 Electroactivation in parthenogenesis Repeated DC square wave pulses can activate oocytes to divide and produce haploid embryos. Diploid cells can be obtained by inhibiting the second polar body by cytochalasin B or by electrofusion. 2.1.2 Preparation of tetraploid embryos The application of direct current pulses at the two-cell stage of the embryo can result in the fusion of cells to generate tetraploid embryos. This has been applied in mice, pigs and cattle [10]. 2.1.3 Preparation of Embryonic Stem Cell Chimeras Chimeric transgenic mice can be prepared by electrotransfection of embryonic stem cells. The stem cells are injected into the recipient's blastocyst and the blastocyst is then transferred into the mother's uterus. Hybrids born between offspring produce homozygotes for reproductive research. Embryonic stem cell chimeras such as mice, pigs, rabbits, and ? have been prepared [11]. 2.2 The application of electrofusion in nuclear transfer technology In 1938, when Hans Spemann proposed the concept of nuclear transfer, cloning technology has begun to emerge. The first cloning in amphibians was reported in 1952, but it was not until 1970 that the frogs were cloned successfully. Nuclear transplantation is currently a hot technology for reproductive research in many species. The purpose of nuclear transfer cloning is to transfer the nuclei of the cloned organisms into the host system to guide the embryonic development and produce new individuals. The needle is first inserted into the zona pellucida of the enucleated oocyte, and the donor cells are injected into the oocyte peripheries of the oocyte. The alternating current provided by the cell fusion apparatus aligns the donor cells and oocytes, and then 1 to several DC waves cause the nuclear transfer embryo to be activated and divide. The embryonic cells resulting from the division are transplanted into the mother's uterus for pregnancy until production. 2.2.1 Electrofusion of the Donor and Recipient Cells Electrofusion The cell membrane is first fused, and then the cells are rounded into one cell. Factors affecting fusion are: cell alignment, fusion buffer, pulse parameters, electrode shape, and oocyte maturation. The first step in the fusion process is cell alignment. In this process the cells are arranged in a specific direction so that the fused cell membrane is perpendicular to the direction of the electric field. This arrangement can be done manually or with an alternating electric field, which can operate multiple embryos at the same time. Fusion buffers are usually lower ionic strength solutions such as mannitol, glucose and sucrose at concentrations between 0.28-0.3M. 10-100mM Ca2+ enhances fusion efficiency [12]. Pulse parameters include electric field strength, pulse time, and pulse number. In the fusion of nuclear-transplanted oocytes, the electric field intensity is usually between 600 V/cm and 3.6 kV/cm, and the pulse time is often between 30-250 ms. Studies have shown that the optimal parameters required for different types of cells vary. In general, the electric field intensity is inversely proportional to the pulse time, and a stronger electric field can compensate for the shorter pulse time [13]. A single pulse can cause cells to fuse. Some scholars believe that increasing the number of pulses can improve the efficiency of fusion, but some people have the opposite opinion. There are not many studies on the electrode. The commonly used columnar electrodes are parallel, and the gap is 0.2-0.5mm. 2.2.2 Electrostimulation of fused cells Activated cells often use DC square wave with different duration. Activation of electrical stimulation during nuclear transfer is also essential and effective for cell fusion. Collas' research shows that the mature oocyte activation efficiency is relatively high. The activation efficiency has nothing to do with the electric field intensity and the pulse time but is related to the pulse waveform. Non-uniform electric field can get higher activation efficiency. The optimal electric field strength depends on the electrode gap. Both the number of pulses and the interval between pulses increase the activation efficiency [14]. 3. Nuclear Transplantation for Different Nuclear Sources 3.1 Embryo-Based Nuclear Transplantation Mammalian embryonic clones produced by nuclear transfer involve fusion between one of the 8-64 cell embryos and the enucleated mature oocytes. Ilmense and Hoppe first reported in 1981 the generation of a nuclear transfer to a living individual [15]. Their research has not been repeated, but it has produced more efficient nuclear transfer technology [16]. Successful nuclear transfer experiments in many species use electrofusion technology. Li Meng and Don Wolf applied this technique to the results of an experimental study of cloned rhesus monkeys published in 1997 by primates [17]. Their work provides an effective animal model for gene therapy. 3.2 Nuclear Transfer Based on Embryonic and Primary Animal Cell Lines There are many limitations to the use of embryonic blastomeres as nuclear donors. How to generate suitable embryonic cells is a bottleneck, and the isolated split spheres are difficult to genetically manipulate in vitro, making it impossible to efficiently transfer genes to them before cloning. Therefore, many scholars are engaged in the development of nuclear transfer strategies based on embryonic or primary animal cell lines. In 1996, the Ian Wilmut research group in Scotland reported that they used cell lines to obtain transgenic sheep [18]. The cell lines used for nuclear transfer were from embryos that had been cultured for 6-13 passages in vitro and were stopped by serum starvation prior to transplantation. In 1997, the use of sheep sheep fetal fibroblasts transfected with human factor VIII gene was successful in obtaining a nuclear-transplant sheep clone [19]. Cibelli, Stice and Robl report for the first time the use of immortalized fetal bovine fibroblasts as a nuclear donor to obtain cloned transgenic cows. Their work demonstrated for the first time that actively dividing cells can continue to develop after nuclear transfer [20]. 3.3 Nuclear transfer studies of nuclear transfer application cell lines based on differentiated mature cells remove obstacles to the cloning of differentiated mature cells. Ian Wilmut reported in 1997 the first animal clone from matured cells, sheep Dolly[21], and the result shocked the entire world. The nucleus provided to Dolly was a mammary epithelial cell of an adult sheep, and the cells were also treated with serum starvation induction. Since then, there have been reports of the use of somatic cells as nucleus donors to obtain cloned animals [22-24], which proves that Dolly's technical route is indeed feasible. 4, the performance of electroporation equipment Early electroporation equipment waveform and time are fixed, inconvenient to use and the effect is poor. The 20 years of development have greatly improved the technical level of the equipment itself and accessories. 4.1 Pulse shape Early exponentially decaying pulses have been used up to now and remain on the design of low-end products, mainly for gene transfection. The newly developed square-wave pulse products have become the mainstream on the market. The purity and reproducibility of the square-wave pulse in many brands are not exactly the same, and the products of those professional manufacturers should be selected. Some products use a series of high-frequency pulses instead of square waves, and the efficiency of the introduction of genes and cell fusion remains to be further confirmed. In addition to direct current pulses, some devices for cell fusion can also generate non-sinusoidal AC waves that align cells in the direction of the electric field and achieve higher fusion efficiency than pure DC pulses. 4.2 Pulse intensity and time Due to the different pulse intensity and time required for the operation of bacteria and cells, the low-end product has a narrow range of strength and time adjustment and therefore has a single role. General-purpose products can generate voltages of 0-3000 V and pulses of 1 microsecond to 10 seconds, so they can be applied to various operations such as cell, bacterial transgene and cell fusion, and embryo engineering. 4.3 Peripherals Cell operating systems with monitors, printers, and remote control interfaces have emerged. The use of these devices in conjunction with these devices allows for easy recording and optimization of experimental parameters and results in the best results in the shortest possible time.
our Cat Snacks supports healthy appetite and digestion,soft,moist texture and delicious,promotes healthy immune system and could feed whole piece or break into small pieces for Cat Treats.Always be sure to provide plenty of water.We suggest that cat snacks is higher moisture and smaller than Dog Snacks,becasue the mouth of cat is small and it can increase the cat's palatability.Beauty Hair,Enhence Immunity and Intestinal Health are all good advantage.
Cat Snacks
Chicken Cube For Cat,Chicken Sandwiches For Cat,Soft Cat Snacks,Soft Cat Food
Eastan Pet , http://www.eastanpet.com