Genetic engineering introduces new genetic material into an organism to study gene function or model diseases. This process uses delivery tools called vectors to carry and insert new genes, or transgenes, into a host’s genome. One specific vector is the Bacterial Artificial Chromosome (BAC), a tool used for precise genetic research.
The Components of BAC Transgenesis
BAC transgenesis involves two components: the transgene and the Bacterial Artificial Chromosome. A transgene is a segment of DNA, often a specific gene, prepared and introduced into an organism’s genome. This allows researchers to introduce a new function, like producing a fluorescent protein, or to study a specific gene version, such as one associated with a human disease.
The Bacterial Artificial Chromosome is the vector that carries the transgene. A BAC is a large, engineered DNA molecule based on a bacterial fertility plasmid (F-plasmid). Its defining characteristic is its capacity to hold large fragments of foreign DNA, up to 300,000 base pairs, distinguishing it from older vectors like plasmids that carry smaller fragments.
The need for a vector that could carry large genetic payloads arose during the Human Genome Project. BACs are maintained in host bacteria, like E. coli, at a low copy number of one or two copies per cell. This low number ensures the stability of the inserted DNA, preventing rearrangements and keeping the large fragment intact during bacterial replication.
The Creation and Delivery Process
Creating a transgenic organism with a BAC starts with modifying the chromosome in bacteria. Scientists use a technique called homologous recombination, or “recombineering,” to precisely insert the desired transgene into the BAC. This method allows for exact placement, such as replacing a mouse gene with its human equivalent. This work is performed inside E. coli cells to produce the modified BACs.
Once engineered, the BAC is isolated from the bacteria. The large size of the BAC makes it susceptible to breaking, so the purified DNA must be handled carefully to remain intact. The most common delivery method for the BAC into the host organism is pronuclear microinjection.
In this procedure, the purified BAC DNA is injected directly into the pronucleus of a fertilized egg, often from a mouse. The injection is performed at a low DNA concentration to optimize the chances of successful integration into the egg’s genome. The injected egg is then transferred into a surrogate mother to be carried to term. The resulting offspring may carry the BAC transgene integrated into their genetic material.
The BAC integrates at a random location within the host’s genome. This nonspecific insertion means offspring can have different numbers of transgene copies at various locations. Scientists screen the offspring to identify “founder” animals that have incorporated the transgene and can pass it to subsequent generations.
Achieving Physiological Gene Expression
The main advantage of using BACs is their ability to facilitate gene expression that mimics the natural state. A gene’s activity is governed by its coding sequence and surrounding DNA known as regulatory elements. These elements can be located far from the gene and dictate when, where, and at what level it is activated.
Because BACs carry large DNA fragments, they can hold the target gene and its complete set of native regulatory elements. This inclusion allows the transgene to be expressed accurately in the correct tissues, at the proper developmental stages, and in response to the right signals. The transgene then behaves much like the organism’s own genes.
This contrasts with earlier methods using smaller vectors like plasmids, which lack the space for all necessary long-range regulatory elements. Consequently, transgenes delivered via plasmids exhibit unpredictable or incorrect expression patterns. They might be expressed in the wrong tissues, at the wrong time, or at incorrect levels, leading to misleading results.
The large size of the BAC also helps insulate the transgene from “position effect variegation.” This phenomenon occurs when a transgene’s expression is influenced by the genomic environment at its random insertion site. By carrying a large buffer of its native genomic context, the BAC transgene is less susceptible to these outside influences, ensuring its expression pattern remains stable.
Applications in Biomedical Research
BAC transgenesis is a useful tool in biomedical research for creating accurate animal models of human genetic diseases. Many human diseases, like Huntington’s or Alzheimer’s, are caused by mutations in large or complexly regulated genes. BACs make it possible to insert the entire human gene and its regulatory machinery into a mouse’s genome, creating a “humanized” model that better reflects the human condition.
These animal models allow researchers to study disease progression, test potential therapies, and investigate underlying molecular mechanisms. For example, scientists have used BACs to create mouse models for seizure disorders by introducing a gene mutation associated with epilepsy in humans. This enables detailed observation of how the gene functions and contributes to the disease.
Another application is studying gene function with reporter genes. Scientists use recombineering to attach a gene that produces a fluorescent protein, like Green Fluorescent Protein (GFP), to a gene of interest within a BAC. When this modified BAC is introduced into an animal, the fluorescent protein is produced wherever the target gene is active. This allows researchers to visually track the cells and tissues expressing that gene, providing a map of its activity. The technique is useful in neuroscience for mapping neural circuits and understanding brain function.