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Although recent advancements in site-specific recombinase systems and site-specific endonucleases have largely contributed to the improvement of targeted transgenesis, a great number of transgenic mice are generated using the conventional random integration method. Different vectors are used for random incorporation of transgenes, including plasmid vectors, bacterial artificial chromosomes (BACs), and retrovirus vectors.
Features of Pronuclear Microinjection
The pronuclear microinjection method was developed over 30 years ago and has been widely used to address a variety of biological questions. Typically, transgenic mice are generated by microinjecting the transgenic constructs, either BACs or plasmids, into fertilized eggs (oocytes or zygotes). Presently, plasmids are the most commonly used vectors because of their versatility and simplicity in use.
An alternative way to effectively introduce a transgene into an egg is the use of a retrovirus vector. The newer retroviral method is highly efficient by has limitations such as high titers of vectors are needed for successful gene transfection; small DNA fragments are accepted by these vectors, and so on. As a result, a great majority of transgenic mice are generated using the pronuclear microinjection method.
Plasmids are convenient and efficient cloning vectors for carrying out a variety of recombinant DNA procedures. However, compared to BACs, they can carry only relatively small DNA fragments and both the transformation efficiency and plasmid stability decrease dramatically when the DNA insert is larger than 25 kb. Moreover, small transgenes are more prone to chromosomal position effects, which often lead to uncontrolled transgene expression. Despite these limitations, plasmid vectors remain the most commonly used transgenic vectors to researchers for they have accumulated years of experience of experimentation and optimization.
Design of Transgenic Construct
A properly constructed transgenic vector is a prerequisite for the successful production of transgenic animals as well as defined expression of the transgene of interest. In general, transgenic constructs must have all the critical elements for gene expression to occur, such as promoters, introns, the protein coding sequence of interest (cDNA or genome) and a poly (A) site. Besides, to release a transgenic cassette from a vector, the construct must be designed with unique restriction enzyme sites flanking the transgenic cassette. Hence small transgenes are prone to influences of the neighboring chromatin environment, genomic boundary elements are employed to reduce chromosomal position effects.
Meanwhile, Creative Biolabs offers the most up to date and comprehensive model creation services besides microinjection methods, please see below for more information:
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