Cre-GFP Adeno-associated virus(AAV Serotype 9)

Glucocorticoids are steroids involved in key physiological processes such as development, metabolism, inflammation, and stress response, and are primarily used as exogenous drugs to treat various inflammatory diseases. They act through glucocorticoid receptors (GR) expressed in most cells. Exogenous glucocorticoids negatively affect the function of Leydig cells in the testis, resulting in reduced androgen production. However, endogenous glucocorticoids are produced by the adrenal glands and testis, but whether their effects on the GR in Leydig cells regulate steroidogenesis is unknown. This study aimed to determine the role of endogenous GR signaling in adult Leydig cells. The researchers developed and compared two models: one driven by an inducible Cre transgene driven by expression of the Cyp17a1 steroidogenic gene (Cyp17-iCre), which depletes GR during development, and another driven by a viral vector (AAV9-Cre), which depletes GR in adulthood. Compared to the Cyp17-iCre model, AAV9-Cre eliminated GR in adult mouse Leydig cells, resulting in more efficient depletion of GR from Leydig cells. Importantly, depletion of GR in adult mouse Leydig cells resulted in reduced expression of the luteinizing hormone receptor (Lhcgr) and steroidogenic enzymes required for normal androgen production. These findings suggest that Leydig cell GR signaling plays a physiological role in the testis and emphasize that a normal balance of glucocorticoid activity in the testis is important for steroidogenesis.

Here, to validate the targeting ability of AAV9-Cre in adult testis, the researchers used a Cre-inducible Tomato Red Floxed reporter line (TRTR) to verify the expression site of the AAV9-Cre transgene (via expression of RFP). TRTR adult (postnatal day 80) males were injected intratesticularly with vector, AAV9-GFP, and AAV9-Cre-GFP (all carrying transgenes downstream of the CMV promoter), and testes were collected 7 days post-injection (dpi) (Figure 1A). Fluorescence was assessed on freshly dissected testes. Compared to vector-injected testes, both AAV9-GFP and AAV9-Cre-GFP induced GFP expression in the interstitium, but only AAV9-Cre-GFP produced RFP expression within the testis (Figure 1B). AAV9-Cre-GFP is a suitable model for acute targeting to the testis with minimal off-target effects. Next, to confirm that the AAV9-induced Cre/LoxP system specifically targets Leydig cells within the testis, the researchers colocalized the Leydig cell markers 3βHSD and RFP. RFP localization was restricted to 3βHSD-positive Leydig cells in the testis (Figure 1C). The number of RFP-positive and RFP-negative Leydig cells was quantified in vector-, AAV9-GFP-, and AAV9-Cre-GFP-injected testes (7 dpi). According to immunohistochemistry (Figure 1C), only AAV9-Cre-GFP induced the expression of RFP in ~97% of Leydig cells (Figure 1D). These data indicate that AAV9-induced Cre/loxP can target Leydig cells within 7 dpi and is therefore a suitable model for inducible knockdown of GR expression in adult mice.

Figure 1. RFP expression in Leydig cells following delivery of the Cre Recombinase using adeno-associated virus serotype 9 (AAV9-Cre). (Gannon A L, et al., 2022)