TCR 1

• The Cre-lox and FLP-FRT systems are two of the most exciting and versatile genetic tools designed in the last 30 years.
• Both the Cre-lox and FLP-FRT systems allow the location and timing of gene expression to be closely regulated.
• Tu et al constructed the pancreatic cancer model of tree shrew using lentivirus and analyzed the gene expression profile by RNA sequencing.
• Compared with non-human primates, the tree shrew has the advantages of small size, short reproductive cycle, easy feeding and so on.
• The results showed that the molecular mechanism of the tree shrew pancreatic cancer model was more similar to that of the human pancreatic cancer model than that of the mouse pancreatic cancer model.
• The tree shrew also allows tree shrews to replace primates for cancer research.
• The Cre-lox system is based on the ability of the P1 bacteriophage cyclization recombination (Cre) recombinase gene (cre) to effect recombination between pairs of loxP sites.
• In a "Cre-lox" mouse, Cre recombinase can either activate or inactivate a gene of interest.
• To use the Cre-lox system, an investigator has to produce a Cre-lox mouse, typically by breeding a Cre mouse to a loxP mouse.
• A Cre mouse contains a Cre recombinase transgene under the direction of a tissue-specific promoter; a loxP mouse contains two loxP sites that flank a genomic segment of interest, the "floxed" locus.
• Depending on the location and orientation of the loxP sites in a Cre-lox mouse, Cre recombinase can initiate deletions, inversions, and translocations of the floxed locus.
• The FLP-FRT system involves using flippase (FLP) recombinase, derived from the yeast Saccharomyces cerevisiae (Sadowski 1995).
• FLP recognizes a pair of FLP recombinase target (FRT) sequences that flank a genomic region of interest.
• Gain-of-function studies are often used to study oncogenes in mouse models.
• The CRISPR/Cas9 system consists of a Cas9 nuclease, which can be directed to any genomic locus by an appropriate single guide RNA (sgRNA).
• DNA transposons have shown great promise in transposon-mediated insertional mutagenesis.
• Retrotransposons, due to their low integration efficiency, are rarely used for the production of transgenic mice.
• Zinc-finger or TALEN nucleases can be engineered to act as a site-specific nuclease, cutting DNA at strictly defined sites, which enables ZNF or TALEN nucleases to target unique sequences within complex genomes.
• Mutagenesis relies on a transposase enzyme, which distinguishes specific DNA sequences and “cuts” the DNA between them.
• Transposons are DNA sequences with the ability to move from one location of the genome to another.
• The two most effective transposons are Sleeping Beauty and PiggyBac.
• Silencing, or better, downregulating gene expression of a target gene by small interfering RNA (siRNA) has been mainly used to study gene function.
• ZNFs and TALENs are produced by combining a DNA-binding domain with a DNA-cleavage domain.
• The excised DNA is then reintegrated at another site in the genome.
• Fluorescent and bioluminescent optical imaging are the most commonly used techniques for visualization due to their increased sensitivity, relative inexpensiveness, and less time-consuming and user-friendly features compared with histological, genetic, or biochemical methods.
• Three kinds of engineered nucleases have been developed and tested for DNA modulation: zinc-finger nuclease (ZNF), transcription activator-like effector (TALEN) nuclease, and the latest clustered regularly interspaced short palindromic repeat (CRISPR)/-associated (Cas9) system.
• RNA interference in mice represents an alternative to knockout mice, or, more accurately, a knockdown mouse.
• Transposon-based insertional mutagenesis can be used in genetic screens to identify novel cancer-causing genes, such as oncogenes or tumor-suppressor genes.
• RNAi does not generate a completely loss-of-function allele.
• Knock-in models of oncogene overexpression can be used to study how the oncogene drives carcinogenesis in vivo.
• The conventional random insertion mouse model can be produced by viral vector-based transfection of early mouse embryos or by pronuclear injection of the transgene directly into fertilized oocytes.
• In the knock-in permissive locus model, a gene of interest inserted into a specific region of the genome using homologous recombination.
• Conditional knock-in models can be generated using tissue-specific promotors or by inserting a strong translational and transcriptional termination (STOP) sequence flanked by loxP or FRT sites between the promotor sequence and the gene of interest.
• To observe the expression of the targeted gene at the transcriptional or translational level, reporter knock-in mouse models can be used.
• Scientists measure the time period that mice find the same platform in the Open Field task, a simple sensorimotor test used to determine general activity levels, gross locomotor activity, and exploration habits in rodent models of CNS disorders.
• Compared with the most commonly used mouse models, the zebrafish model has some unique advantages in cancer research: small size, low cost and fast reproduction; transparent embryos, it is convenient to observe and track the proliferation, spread and metastasis of cancer cells in real time; transgenic zebrafish and immunodeficient zebrafish can remain transparent after adulthood.
• Most of the animal models commonly used in cancer research are small animal models, such as mice, rats, zebrafish, fruit flies and so on.
• Small animal models have many advantages, such as strong reproductive ability, low cost, easy maintenance and so on.
• Due to the high cost of breeding and feeding, complex experimental techniques and ethical problems, the use of non-human primates has been limited.
• Among them, mice and zebrafish are the most widely used.