We all know someone with cancer, obesity, or heart disease. If not, then you know someone who is aging, and you, yourself are experiencing this phenomenon. What do these have in common?
These are all research areas that use transgenic knockout animals. Researchers can study the function of a gene by removing it from an organism and studying that animal’s phenotype. One way to remove a gene is with Cre-lox recombination. If the genome were a bookshelf and the books were a gene sequence, then loxP, shown in blue, would be the bookends that you set around books to be donated, or the gene to be deleted. When the Cre-recombinase enzyme recognizes loxP, it snips out the intervening sequence, like you donating books. This is a common procedure that has been used in labs around the world since the 1980s. But what if it has flaws?
Brain-derived neurotrophic factor, or BDNF, is a protein that helps synapses grow and function. We study it because it’s involved with ALS, which many remember from the Ice Bucket Challenge. When we added LoxP sites around the BDNF allele, animal handlers noted unusual behaviors and phenotypes in these mice. As evidenced by this photo, mice with LoxP can grow significantly larger than controls. These mice make us ask: is LoxP really a “benign” insert, as was previously believed? And if not, does that call into question all of the data that we’ve gathered from labs studying BDNF?
It has been theorized that small gene inserts like LoxP, neo cassettes, and FRT sites may affect local genes, but no studies have been conducted on this phenomenon and no current literature (2024) has documented this interference (Meier et al., 2010). Although the creators of this transgenic line originally described the BDNF-floxed mice as physiologically normal and fertile, numerous tests have contradicted this claim (Rios et al., 2001). BDNF-floxed mice are hyperactive, show high rates of intermale aggressiveness, and display agitation when handled by four weeks of age (Brandt, 2018). Other researchers have noted increased rates of infanticide, stereotypy behaviors like route tracing and somersaulting, and hyperphagia (Clow & Jasmin, 2010). This leads us to believe that the loxP sites cause unprecedented interference with gene expression in BDNF-floxed mice.
One way to assess the viability of these mice is with a prepulse inhibition test. Prepulse inhibition occurs when a loud, startle pulse follows a soft, prepulse. “Normal” brains are so good at gating out unnecessary information that they react less to the second pulse because the brain realizes: “Oh, that’s just noise.” Our mice overreact, which is an endophenotype associated with human conditions like OCD, Tourette’s, and schizophrenia. We can then look at the neural structures underlying these behaviors, the cortico-striato-pallido-pontine circuit (CSPP), to assess for physiological variation.
First, we will stain neural tissue with thionine. Thionine binds to nissle substances, the RNA and proteins packed into the endoplasmic reticulum of neurons. This stain will allow us to visualize individual cell density and distribution (Eichler & Taylor, 1976). Are neural structures in floxed-BDNF mice the same as in WT? Is there a difference in cell density? Finally, is the percentage of white matter the same as in WT animals, or altered, as is sometimes associated with deficit PPI testing (Kumari et al., 2008)?
After assessing structure morphology, we’ll assess BDNF mRNA distribution with a BDNF mRNA-specific digoxigenin probe to help us determine if the loxP gene insert affects local gene expression. Abnormal distribution of BDNF will be used to validate further genetic testing. Future research should focus on gene site assessment. Perhaps one or both of the LoxP sites were inserted into an existing gene, disrupting its function. Alternatively, a deletion or mutation in the genome may have occurred when the transgenic line was created. Further still, the physical gene structure may have brought sequence elements into contact which would otherwise be separate, overexpressing or preventing normal gene expression. Analysis in the future should include local gene sequencing, RNA sequencing to determine gene expression, or electron microscopy to assess for abnormal chromatin distribution.
It’s integral not only for the results of these studies but for the millions of animal models that give their lives to science each year that our transgenic technology is fully operational. That way we don’t accidentally donate something we meant to keep, or put our copy of “The Hobbit” in with Lemony Snicket. That would be a Series of Unfortunate Events.
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