BAC transgenic mice are laboratory mice that carry a segment of foreign DNA within their genome, introduced using a Bacterial Artificial Chromosome (BAC). A BAC is a large, engineered DNA molecule that can hold substantial pieces of genetic material. These mice are created to introduce new genes, often human genes, into a living animal system. This allows researchers to study how a specific gene functions or contributes to a disease in a whole-organism context.
Advantages of Using Bacterial Artificial Chromosomes
Bacterial Artificial Chromosomes are favored for creating transgenic mice because their properties overcome the limitations of other methods. Genetic vectors are tools used to carry foreign DNA into an organism, and while smaller circular DNA molecules called plasmids are common, their size is restrictive. BACs were developed to handle much larger genetic sequences, which changed how scientists could study complex genes.
A BAC’s most notable feature is its ability to carry large DNA fragments, ranging from 100,000 to 300,000 base pairs. This capacity allows an entire gene, or even a cluster of related genes, to be included in a single BAC. For long human genes, this capability is necessary for the gene to be transferred completely.
This large capacity also allows for the inclusion of extensive regulatory sequences along with the gene. A gene’s activity is controlled by other DNA sequences known as promoters and enhancers, which can be located far from the gene they regulate. By accommodating such large DNA fragments, a BAC can contain these distant but important regulatory elements, ensuring the genetic instructions are kept intact.
As a result, the transgene is more likely to be expressed in a manner that mirrors the natural gene’s activity. This leads to gene expression at physiological levels with the correct developmental timing and pattern. The resulting mouse model is therefore a more accurate and reliable representation of the gene’s function in a living system.
Creating a BAC Transgenic Mouse
Producing a BAC transgenic mouse is a precise, multi-step laboratory process. The gene of interest, often a human gene, is first inserted into the BAC vector within bacteria like E. coli. These bacteria then replicate, making many copies of the BAC, which are later isolated and purified to ensure only high-quality DNA is used.
The core technique is pronuclear microinjection. This involves using a microscope and a very fine, hollow needle to inject the purified BAC DNA directly into the pronucleus of a fertilized mouse egg. The pronucleus is a structure containing the genetic material from one parent before it fuses with the other’s. This direct injection is an effective way to generate BAC transgenic mice.
The injected embryos are then transferred into a surrogate mother to develop. The recipient female mouse is made “pseudopregnant,” a hormonally induced state where her body is prepared to accept and gestate embryos. The microinjected eggs are surgically implanted into her reproductive tract, where they will hopefully implant and develop into pups.
After birth, the offspring are screened to identify which ones have successfully integrated the BAC transgene into their genome. This is done by taking a small tissue sample, like a tail tip, and using Polymerase Chain Reaction (PCR) to detect the foreign DNA. Mice that test positive are known as “founder” mice and can be bred to establish a stable line of transgenic animals for research.
Applications in Scientific Research
BAC transgenic mice are a valuable tool in biomedical research. Their primary use is creating animal models for human genetic diseases. By introducing a human gene associated with a disorder into a mouse, researchers can replicate aspects of the human condition, allowing them to investigate disease mechanisms and test potential therapies.
These models are useful for diseases caused by mutations in large or structurally complex genes. For instance, Huntington’s disease is caused by a mutation in the large human huntingtin (HTT) gene. A BAC can carry the entire human HTT gene with the disease-causing mutation, which can then be expressed in the mouse to study how it leads to neuronal damage. Models for Alzheimer’s disease and some seizure disorders have also been developed using this approach.
Beyond modeling specific diseases, BAC transgenic mice are used to explore gene function and regulation. Researchers can attach a reporter gene, such as one that produces a green fluorescent protein (GFP), to the gene of interest within the BAC. When the mouse is created, any cell that expresses the target gene will also glow green, providing a visual map of where and when a gene is active.
Another use for these models is in “rescue” experiments. A researcher might start with a knockout mouse, which has had a specific gene deleted, often resulting in a defect. By introducing a healthy copy of that gene back into the knockout mouse using a BAC, scientists can see if the normal function is restored. If the symptoms are reversed, it provides strong evidence that the deleted gene was responsible for the observed defects.
Key Considerations and Limitations
The creation and use of BAC transgenic mice involve several technical challenges. A significant issue is that the BAC DNA integrates into the mouse genome randomly. If the BAC inserts itself into the middle of a functional mouse gene, it can disrupt that gene’s activity and create an unexpected new trait, complicating the interpretation of results.
Another issue is the variation in the number of transgene copies that integrate into the genome. The microinjection process does not control how many copies of the BAC are incorporated. If a large number of copies are integrated, it can lead to the overexpression of the target gene at levels far beyond what is natural, producing effects that are artifacts of the high dosage.
The process is technically demanding and has a low efficiency rate. Preparing the large BAC DNA requires great care, as the molecules are fragile and can break during purification or injection. If the BAC DNA fragments, an incomplete version of the transgene may be integrated, which may not function as expected.
Generating and validating these transgenic lines requires significant time and resources. Researchers must characterize the mice to ensure observed traits are due to the transgene’s expression and not an artifact of the method.