Alzheimer’s disease (AD) affects over 5 million individuals each year in the United States. Furthermore, most cases of AD are sporadic, making it extremely difficult to model and study in vitro. CRISPR/Cas9 and base editing technologies have been of recent interest because of their ability to create single nucleotide edits at nearly any genomic sequence using a Cas9 protein and a guide RNA (sgRNA). Currently, there is no available phenotype to differentiate edited cells from unedited cells. Past research has employed fluorescent proteins bound to Cas9 proteins to attempt to enrich for edited cells, however, these methods are only reporters of transfection (RoT) and are no indicative of actual base-editing occurring. Thus, this study proposes a transient reporter for editing enrichment (TREE) and Cas9-mediated adenosine TREE (CasMasTREE) which use plasmids to co-transfect with CRISPR/Cas9 technologies to serve as an indicator of base-editing. Specifically, TREE features a blue fluorescent protein (BFP) mutant that, upon a C-T conversion, changes the emission spectrum to a green fluorescent protein (GFP). CasMasTREE features a mCherry and GFP protein separated by a stop codon which can be negated using an A-G conversion. By employing a sgRNA that targets one of the TREE plasmids and at least one genomic site, cells can be sorted for GFP(+) cells. Using these methods, base-edited isogenic hiPSC line generation using TREE (BIG-TREE) was created to generate isogenic hiPSC lines with AD-relevant edits. For example, BIG-TREE demonstrates the capability of converting Apolipoprotein E (APOE), a gene associated with AD-risk development, wildtype (3/3) into another isoform, APOE2/2, to create isogenic hiPSC lines. The capabilities of TREE are vast and can be applied to generate various models of diseases with specific genomic edits.

Cocaine is a powerful psychomotor stimulant that can affect serotonin (5HT), dopamine, and norepinephrine systems in the brain. Previous studies with 5HT1B receptor agonist, CP94253, have shown dose-dependent decreases in cocaine-self administration in male rats during maintenance. However, these studies do not take into consideration sex differences between male rats and female rats. Female rats introduce a new complexity because they constantly undergo an estrous cycle that consists of four phases, metestrus, diestrus, proestrus, and estrus. It was hypothesized that cocaine infusions and active lever response rates would greatly decrease during proestrus and estrus in comparison to metestrus and diestrus due to hormonal level differences of LH, FSH, progesterone, and estradiol. In this study, female rats were trained to self-administer a training dose of 0.75 mg/kg/infusion on a fixed progressive ratio (FR5). Rats were then pretreated with CP94253 to test the effects of this 5HT1B agonist on female rat cocaine self-administration during the estrous cycle. Results showed there was no three-way interaction between cycle phase, pretreatment, and cocaine dose on infusions or active lever responses. However, pretreatment with CP94253 decreased cocaine intake and active lever responses at high cocaine doses, regardless of cycle phase. Lastly, there was a two-way interaction between pretreatment and cycle phase in which active lever responses decreased during diestrus and proestrus. These results imply that CP94253 enhances cocaine's effect regardless of cycle phase. Future work can work with ovariectomized (OVX) female rats to observe cocaine self-administration during controlled cycle phases.