Today's technology recommended gene knockout technology

**1. Overview:** Gene knockout is a modern molecular biology technique that has been developed since the late 1980s. It involves inactivating or deleting specific genes in an organism through various methods. The most common approach is based on DNA homologous recombination, where a target gene is replaced by a designed homologous fragment to achieve gene inactivation. Over time, new techniques such as gene insertion mutations and RNA interference (RNAi) have also been applied to achieve similar outcomes. These advancements have made gene knockout more versatile and efficient. **2. Various Principles and Methods for Gene Knockout:** **2.1. Gene Knockout Using Homologous Recombination** Homologous recombination-based gene knockout was first developed in the mid-1980s. The isolation of embryonic stem (ES) cells in the early 1980s and their successful in vitro culture provided the technical foundation for this method. In 1985, the discovery of homologous recombination in mammalian cells laid the theoretical basis for gene knockout. By 1987, Thompson successfully created the first complete mouse model using ES cell knockout. Today, this method remains the most widely used for generating gene knockout animal models. **2.1.1 Basic Steps for Constructing a Gene Knockout Animal Model via Homologous Recombination (Figure 1):** - **Vector Construction**: A recombinant vector carrying a marker gene (e.g., neo or TK) is constructed by replacing the target gene with a homologous fragment. - **ES Cell Acquisition**: Embryonic stem cells, especially from mice, are commonly used. Other species like rabbits, pigs, and chickens are also employed. - **Homologous Recombination**: The recombinant vector is introduced into ES cells using electroporation or microinjection. This allows the foreign DNA to integrate into the genome. - **Screening**: Due to low recombination efficiency, screening methods like PNS (positive-negative selection) and PCR are used to identify correctly modified cells. - **Phenotypic Analysis**: Observing changes in the mouse’s phenotype helps understand the function of the knocked-out gene. - **Homozygous Generation**: At least two generations are needed to obtain stable homozygous knockout mice. **2.1.2 Conditional Gene Knockout** Conditional gene knockout allows for spatial and temporal control of gene inactivation. It uses the Cre/LoxP system to create a "switch" that enables targeted gene modification. When Cre recombinase is expressed, it excises the gene flanked by LoxP sites. This method avoids lethal effects caused by global gene deletion and is useful for studying tissue-specific functions. **2.1.2.1 Inducible Gene Knockout** This method builds on the Cre/LoxP system but introduces inducibility through external factors like tetracycline or hormones. This allows researchers to control when and where gene knockout occurs, avoiding developmental issues. **2.2. Gene Knockout Using Random Insertion Mutations** This technique relies on vectors like viruses or transposons to randomly insert mutations into the genome. A library of cells with random insertions is then screened to identify those with the desired gene knockout. This method is particularly useful for large-scale functional studies. **2.2.1 Gene Capture Method** The gene capture method is a newer approach that uses vectors containing a reporter gene (like neo) to identify cells with random insertions. This simplifies the identification of target genes and reduces the need for extensive cloning. **2.2.2 Advantages and Disadvantages** While gene capture saves time and resources, it only targets genes expressed in ES cells, limiting its scope. It also lacks precision in genetic modifications compared to homologous recombination. **2.3. RNAi-Mediated Gene Knockout** RNA interference (RNAi) is a powerful tool for gene silencing. Double-stranded RNA triggers the production of siRNAs, which guide the degradation of target mRNA. RNAi is simpler and faster than traditional methods and is widely used in studying gene function, especially in mammalian systems. **2.4. Other Techniques** Other emerging techniques include TFOs (Triple Helix Forming Oligonucleotides) and antisense technology. These methods are still under development but show promise for future applications. **3. Applications and Future Prospects of Gene Knockout Technology** - **Biological Models**: Gene knockout models help study gene function, metabolic pathways, and disease mechanisms. - **Disease Research and Therapy**: Understanding gene roles aids in developing targeted therapies. - **Xenograft Organs**: Knocking out immune-related genes in animals may enable safe organ transplantation in humans. - **Immunology**: Producing human antibodies in animals could revolutionize drug development. - **Organism Transformation**: Gene knockout supports the creation of genetically modified organisms for agriculture and biotechnology. **4. Limitations of Gene Knockout Technology** Despite its advantages, gene knockout has limitations. Some genes are functionally redundant, making phenotypes hard to detect. Essential genes may cause lethality when knocked out, preventing further study. **References** [1] Wang Youhong. Application Status and Development Prospect of Gene Knockout Technology. Foreign Medicine, 1999. [2] Muller, U. Ten years of gene targeting: targeted mouse mutants, from vector design to phenotype analysis. Mech. Dev. 1999. [3] Ledermann, B. Embryonic stem cells and gene targeting. Exp. Physiol. 2000. [4] Hallmann, A. et al. Gene replacement by homologous recombination in Volvox carteri. Proc. Natl. Acad. Sci. USA, 1997. [5] Thomas, P. Homologous recombination in human ES cells. Nature, 2003. [6] Liu, D. et al. Gene knockout physiology science progress, 1995. [7] Nelson, R.J. et al. Behavior in mice with targeted disruption of single genes. Neurosci Biobehav Rev, 1998. [8] Holmes, A. Targeted gene mutation approaches to the study of anxiety-like behavior in mice. Neuroscience and Biobehavioral Reviews, 2001. [9] Hasty, P. et al. Nature, 1991. [10] Yunfengli. Latest developments in gene knockout technology. Lilac Garden Electronic Journal, 2003. [11] Chen, Q. et al. Gene knockout technique in plant functional group research. Plant Physiology Newsletter, 2004. [12] Klinner, U. et al.

Night Stand

The bedroom is meant to be one of the most comfortable rooms in the house. If you feel like there just needs to be one more addition to your room, you may want to consider a new bedside Table. Nightstands offer you much more than just another piece of furniture; they offer a great storage place for gloves, books, alarm clocks and lamps.

With its sleek and simple designs of one shelf and one storage drawer, you have plenty of choices with how to use it. Nightstands deliver great versatility for whatever your needs are.

Night Stand,Small Night Stand,White Night Stand,Bedroom Night Stands

Shenzhen Guang Disen Furniture co.,ltd , https://www.gdisenfurniture.com