Given that the Cre-loxP system is the wide-usage and powerful strategy for conditional gene knockout mice, we applied it for Brg1gene as the model, with the Mx1-Cre for HSC to construct the conditional Brg1(Brg1fl/flMx1-Cre) (Brg1-CKO) and Alkbh5 knockout (Alkbh5fl/flMx1-Cre) (Alkbh5-CKO) mice. To optimize the knockout condition, based on both knockout rate and survival of CKO mice, we explored the different strategies, different components of poly(I: C), various injection doses and injection schedules.
The poly(I: C) components influences the knockout of Brg1-CKO mice.
To investigate the influence of poly(I: C) components, we firstly constructed Brg1fl/flMx1-Cre mice by injecting two kinds of poly(I: C), and injected 10µg/g poly(I: C) containing free nucleotides every second day for a total of 3 intraperitoneal injections (Fig. 1A), and analyzed the mice 15 days post-injection. The data showed that there was an increasing trend of the body weight in mice, though there was no significant difference in Brg1-CKO mice before and after poly(I: C) injection, as well as for the Brg1fl/fl mice. The body weight of the two groups was increased significantly when compared with that before the injection after poly(I: C) injection for 15 days (Fig. 1B). However, only the Brg1-CKO mice were died after poly(I: C) injection, without significant difference in the survival rate of the two groups (Fig. 1C). The results of q-PCR and WB showed that the expression of Brg1 was significantly lower in samples from Brg1-CKO mice than in those from Brg1fl/fl mice, and a small portion of Brg1-CKO mouse samples slightly decreased Brg1 expression (Fig. 1D, 1E). Consistently, histological analysis showed that naive cells were increased slightly in the BM of the Brg1-CKO mice (Fig. 1F), implying that poly(I: C) with other components could induce the incomplete loss of Brg1 expression.
The poly(I: C) without other components affects the mortality of Brg1-CKO mice.
To reduce the expression of Brg1 more efficiently, we used poly(I: C) without other components, and the injection schedule and dosage had not been changed (Fig. 2A). Although the body weight of control mice was no significant difference before and after poly(I: C) injection, the Brg1-CKO mice showed a prominent reduction of body weight (Fig. 2B). At the time of mice sacrificed, we found an increased body weight of Brg1fl/fl mice, whereas Brg1-CKO mice were severely reduced (Fig. 2B). Mice derived from Brg1-CKO treated with poly(I: C) showed a significant survival disadvantage (Fig. 2C). The expression of Brg1 was found to be decreased in the Brg1-CKO mice, which was compared with Brg1fl/fl mice (Fig. 2D). Western blot analysis showed complete loss of Brg1 in poly(I: C)-treated Brg1-CKO mice (Fig. 2E). Histological analysis of BM in Brg1-CKO mice revealed the enhanced naive cells (Fig. 2F). Collectively, these results suggest that the expression of Brg1was effectively knock out, but the mortality rate of mice was high.
Multiple low-dose poly(I: C) injections affect the rate of knockout and survival in Brg1-CKO mice.
To improve the survival, we adopted the original injection schedule and changed the injection dose from 10µg to 6µg poly(I: C) per gram of body weight (Fig. 3A). In terms of body weight, we found that the two groups of mice also remained unaffected before and after poly(I: C) injection. The weight of control mice was increased significantly on day 15 more than that of mice before poly(I: C) injection, while that of Brg1-CKO mice was lost severely (Fig. 3B). Although Brg1-CKO mice treated with poly(I: C) showed a shorter survival time than that from Brg1fl/fl mice, the survival rate of the former can reach 50% (Fig. 3C). Notably, both the results of qPCR and WB showed a complete loss of Brg1 expression in poly(I: C)-treated Brg1-CKO mice (Fig. 3D, 3E). We also observed more naive cells in the BM of the Brg1-CKO mice (Fig. 3F). Taken together, these results suggest that knocking out Brg1 expression and improving the survival of mice could be effective when reducing the poly(I: C) injection dose with multiple injection times.
A single high-dose poly(I: C) injection will reduce knockout efficiency in Brg1-CKO mice.
Considering that a single high-dose injection of poly(I: C) can also knock down target genes efficiently [17], we implemented it as the scheme (Fig. 4A). On the 4th day of poly(I: C)-induced knockout, two groups of mice had a normal weight, whereas all mice had a significant increase on the 15th day (Fig. 4B). In addition, we found that Brg1-CKO mice had died partially, and significant differences were observed (Fig. 4C). By contrast, both the results of qPCR and WB analysis indicated that Brg1 expression was incompletely lost in Brg1-CKO mice (Fig. 4D-4E). The naive cells in the BM of Brg1-CKO mice did not change significantly (Fig. 4F), indicating that a single high-dose of poly(I: C) is not suitable for Brg1-CKO mice.
Multiple media or low-dose poly(I: C) injections can ensure complete depletion of Alkbh5 mice with non-lethal phenotypes.
To rule out that this drug injection protocol is only applicable to brg1 mice, we use Alkbh5fl/fl-Mx1-Cre mice to verify these three injection protocols. We found that both the results of qPCR and WB showed Alkbh5 can be knocked out completely with 10µg poly(I: C) per gram of body weight injection protocol (Fig. 5A-5B), and Alkbh5 can also be completely knocked out with 6µg poly(I: C) per gram of body weight injection protocol (Fig. 5C-5D). Consistent with the results of previous studies, Alkbh5 can only be partially knocked out with a single high-dose of poly(I: C) (Fig. 5E-5F). Taken together, these results suggest that to ensure that the expression of the target gene is completely exhausted, 10µg or 6µg poly(I: C) per gram of body weight injection protocol can be used.
Before the mice were sacrificed, observe the weight gain of the mice of all injection protocols (Supplementary Fig. 1), but no weight change of the mice was observed before and after 10µg or 6µg poly(I: C) per gram of body weight injection protocol (Supplementary Fig. 1A, 1C), and the weight gain of the mice was observed before and after poly(I: C) per gram of body weight injection protocol (Supplementary Fig. 1B).