Cre-Lox Neurogenetics: 20 Years of Versatile Applications in Brain Research and Counting…

Abstract

Defining and manipulating specific neurons in the brain has garnered enormous interest in recent years, because such an approach is now widely recognized as crucial for deepening our understanding of how the brain works. When I started exploring the Cre-loxP recombination for brain research in the early 1990s, it was written off as a dead-end project by a young fool. Yet over the past 20 years, Cre-lox recombination-mediated neurogenetics has emerged as one of the most powerful and versatile technology platforms for cell-specific gene knockouts, transgenic overexpression, Brainbow imaging, neural pathway tracing with retrovirus and CLARITY, chemical genetics, and optogenetics. Its popularity and greater utility in neuroscience research is also largely thanks to the NIH's bold Blueprint for Neuroscience Research Initiative to launch several Cre-driver resource projects, as well as individual laboratories and private research organizations. With newly-discovered, genetically-encoded molecules that are capable of responding to sonar and magnetic stimulation, for sonogenetics or magnetogenetics, respectively, or detecting rapid voltage changes in neurons, Cre-lox neurogenetics will continue to aid brain research for years to come.

Keywords: Cre recombinase; Cre-loxP; brainbow; cognition; learning and memory; mouse brain; neural circuits; optogenetics.

FIGURE 1

DNA recombination involves complex topological and biochemical during DNA replication. DNA recombination is linked with DNA replication during cell divisions in order to exchange genetic materials for the selection of favorable mutations or the elimination of unfavorable or deleterious mutations. Recombination also comes with DNA injury and is therefore tightly coupled with the DNA repair mechanism. Nearly all neurons in the adult brain are post-mitotic to ensure their stability. This explains why brain tumors are in glial cells but not in neurons.

FIGURE 2

Cre-LoxP neurogenetics for achieving region- and cell-type specific analysis of relationships of genes, circuits, and functions in the brain. (A) Strategy to detect the Cre-LoxP recombination in the brain. It required the production of two different transgenic mice: Tg-Cre and Tg-Reporter lines, respectively. These two lines were then crossed to generate the double transgenic mice. CA1-specific recombination was detected after P19 by LacZ method. It is known that CA1 pyramidal cells undergo neurogenesis between E10 and E18 and enter the post-mitotic state by P0. They are well differentiated by P7, with fully established synaptic connections. We have found that Cre-loxP recombination occurs during the middle or end of the third postnatal week in the CA1 pyramidal cells. LacZ in the CA1 pyramidal cells was detected as deep blue color on Nissl stain (in purple-blue) background. (B) Conditional knockout of NR1 gene in a specific cell type and region. The exon 11-21 encoding the entire transmembrane domain and C-terminus were flanked by the loxP (termed floxed) in the introns. The second loxP was followed with the neo cassette gene, which allowed for targeted ES cells selection. Luckily, insertion of loxP sites and the neo gene did not alter NR1 gene expression in the floxed homozygous mice.