A Mitochondrial/Nuclear Exchanged Mouse Strain for Improved Transgenesis and Mutagenesis via Pronuclear Injection
This invention combines the strengths of two commonly used mouse strains (C57BL/6J and FVB/N) to obtain a preferable nuclear genome amenable to pronuclear injection for mouse transgenesis.
Mouse transgenesis has been used extensively for in vivo analysis of gene function and the generation of human disease models. It is a relatively cost-intensive and time-consuming process, requiring approximately one year from establishing the necessary DNA constructs for pronuclear injection to establishing a new transgenic mouse strain with some phenotypic characterization. Microinjection of DNA into zygotic pronuclei remains the predominant methodology to produce transgenic mice, however there is wide variability in the level and pattern of transgene expression, which greatly influences the phenotype. This is due to the random nature of copy number and configuration/integration site of the transgene. Although single-copy transgenesis in mice can be achieved with retroviruses and transposons, these approaches integrate transgenes throughout the genome. As a result, the transgenes are subjected to the local chromatin environment, which can cause endogenous gene disruption.
The C57BL/6 mouse strain is currently the gold standard for phenotypic analysis for many research areas and is the reference strain for the mouse genome sequence, as it is a permissive background for maximal expression of most mutations. However, it is unsuitable for most genetic modification procedures, as it produces zygotes, which are difficult to use for pronuclear injection, a commonly used method for transgenesis and mutagenesis. Undesirable characteristics include poor visualization of pronuclei, granular cytoplasmic matter that extrudes from the embryo after injection, and poor embryonic survival after the injection. Although the FVB/N mouse strain is the preferred strain for pronuclear injections, it has undesirable phenotypes in adult mice including a strong propensity for neurodegeneration.
The use of transgenic mouse models is used extensively by universities and the pharmaceutical industry for in vivo analysis of gene function and generation of human disease models, commonly used for therapeutic preclinical testing. More than any other genetically engineered research animal, mice close closely resemble human biology in their pathophysiology, cellular structure, and organization, with 99% of their genes shared with humans. They also take a leading position as a result of the completion of mouse genome sequencing, expanding collection of relevant gene technology, and biological conservatism.
The technology presents a unique strategy for enhancing pronuclear injection of genetic alterations in the C57BL/6 genetic background, significantly reducing the cost and time associated with the introduction of transgenes and mutations into the C57BL/6 genetic background.
Why it is better:
The individual strengths of both the C57BL/6 and FVB/N mouse strains are combined to enhance pronuclear injection of genetic alterations, significantly reducing the cost and time associated with the introduction of transgenes, while also providing a rapid alternative to backcrossing of transgenes to prevent nuclear crossovers/combinatorial effects.
C57BL/6J mice are used in a wide variety of research areas including cardiovascular biology, developmental biology, diabetes and obesity, genetics, immunology, neurobiology, and sensorineural research. The technology also can be used in metabolic research, as the 6J substrain is most commonly used background strain for the obl ob mouse, which is susceptible to diabetes and diet-induced obesity. The C57BL/6 strain also shows an increased preference for narcotics and alcohol, so is also preferentially used for genetic studies of substance abuse. Mitochondrial-exchange mice also allow direct, unambiguous assessment of mtDNA contributions to disease since there is no complexity introduced by potential nuclear cross-over and combinational effects in the filial generations associated with standard backcrossing methods used to generate conplastic mice.