在Rosa26基因位點(diǎn)定點(diǎn)插入CAG promoter-loxp-stop-loxp-mCherry-EGFP-Map1lc3a-WPRE-polyA表達(dá)框。Map1lc3a也就是LC3，是一個(gè)廣泛表達(dá)的自噬小泡特異標(biāo)志物。該基因Rosa26位點(diǎn)條件性過表達(dá)雜合子小鼠無明顯異常，loxp-stop-loxp表達(dá)框的存在阻止了下游目的基因LC3的轉(zhuǎn)錄，與Cre小鼠交配后，在其后代雙陽性小鼠中，Cre表達(dá)的組織和細(xì)胞類型，loxp-stop-loxp表達(dá)框?qū)⒈磺贸?，目的基因LC3在CAG啟動(dòng)子的驅(qū)動(dòng)下，在缺血性損傷后的吞噬細(xì)胞中以pH依賴性方式表達(dá)mCherry和EGFP。兩個(gè)共同表達(dá)的熒光信號(hào)根據(jù)自噬小泡在細(xì)胞內(nèi)的酸性環(huán)境變化而變化。mCherry在酸性環(huán)境（pKa 4.5）為穩(wěn)定，而EGFP在溶酶體內(nèi)的酸性環(huán)境(pKa 5.9)中會(huì)發(fā)生淬滅現(xiàn)象。這一特性使得細(xì)胞自噬現(xiàn)象根據(jù)細(xì)胞自身溶酶體工作與否區(qū)分開來。在pH較高的自噬小泡中，GFP與mCherry的熒光疊加呈現(xiàn)黃色熒光；而在pH較低的溶酶體中，EGFP淬滅，只能檢測(cè)到紅色熒光信號(hào)?？捎糜跇?biāo)記及追蹤LC3、研究缺血性損傷后各種組織中自噬的起源、進(jìn)展和消失。

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Fig1. Detection of EGFP and RFP fluorescence intensity in brain, heart and liver tissues of R26-CAG-mCherry-EGFP-LC3^+/-, DPPA3-Cre^+/- mice. Starvation(+): The mice were fasted for 48 hours; Starvation(-): The mice were fed normally.

Fig2. Detection of EGFP and RFP puncta in liver tissue sections of R26-CAG-mCherry-EGFP-LC3+/-, DPPA3-Cre+/-?mice. Starvation for 48 h increased the ratio of RFP/EGFP fluorescence expression in liver tissue of R26-CAG-mCherry-EGFP-LC3+/-, DPPA3-Cre+/-??mice. These results suggested there was increased fusion of?autophagosomes with lysosomes after 48h starvation. Starvation(+): The mice were fasted for 48 hours; Starvation(-): The?mice were fed normally.