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Wes全自動(dòng)蛋白質(zhì)表達(dá)定量分析系統(tǒng)應(yīng)用于微量腦組織蛋白表達(dá)分析
點(diǎn)擊次數(shù)：95 發(fā)布日期：2019-3-18
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中腦多巴胺能神經(jīng)元與帕金森、藥物濫用密切相關(guān)
帕金森病和藥物濫用障礙都與中腦多巴胺能神經(jīng)元的功能改變有關(guān)。在外側(cè)中腦，黑質(zhì)(SN)的多巴胺能神經(jīng)元(SN)向背側(cè)紋狀體發(fā)出投射，參與控制運(yùn)動(dòng)，而位于腹側(cè)被蓋區(qū)(VTA)的多巴胺能神經(jīng)元(VTA)支配腹側(cè)紋狀體(伏隔核)和前額葉皮質(zhì)，以參與激勵(lì)和獎(jiǎng)勵(lì)方式。因此，在疾病進(jìn)展的不同階段改變中腦多巴胺能神經(jīng)元的遺傳組成的能力，可以揭示基因在相關(guān)病理表現(xiàn)中的作用，從而促進(jìn)新的治療方法的發(fā)展。
特定基因敲除建立中腦多巴胺能神經(jīng)元研究模型
美國(guó)NIH下屬美國(guó)國(guó)家藥物濫用研究中心（National Institute on Drug Abuse）的多個(gè)研究所，聯(lián)合在神經(jīng)生物學(xué)頂級(jí)雜志Neuron發(fā)表研究結(jié)果：Neuron-Specific Genome Modification in the Adult Rat Brain Using CRISPR-Cas9 Transgenic Rats，Neuron (2019), doi.org/10.1016/j.neuron.2019.01.035 通過(guò)CRISPR-Cas9技術(shù)建立特定神經(jīng)元敲除大鼠模型。
研究摘要
從歷史上看，大鼠一直是行為研究的首選動(dòng)物模型。然而，基因組修飾方面的限制造成了與現(xiàn)有轉(zhuǎn)基因小鼠相比，它們的使用滯后。本文，科學(xué)家們開(kāi)發(fā)了幾種轉(zhuǎn)基因工具，包括病毒載體和轉(zhuǎn)基因大鼠，利用CRISPR-Cas9技術(shù)對(duì)特定成年大鼠神經(jīng)元進(jìn)行靶向基因組修飾。從野生型大鼠開(kāi)始，利用腺相關(guān)病毒(AAV)表達(dá)Cas9或?qū)騌NA(GRNAs)的載體，實(shí)現(xiàn)酪氨酸羥化酶的敲除。隨后構(gòu)建了一個(gè)AAV載體，用于Cre依賴(lài)的gRNA表達(dá)，以及三個(gè)新的轉(zhuǎn)基因大鼠系，將CRISPR-Cas9組分靶向到多巴胺能神經(jīng)元。一只大鼠代表第一只以精原干細(xì)胞為靶點(diǎn)的生殖細(xì)胞基因敲除大鼠模型。本文所描述的大鼠是一種通用的平臺(tái)，用于在成年大鼠的大腦和其他可能表達(dá)cre的組織中進(jìn)行細(xì)胞特異性和序列特異性基因組修飾。
Wes在本文中被用于微量中腦組織和原代神經(jīng)元細(xì)胞蛋白質(zhì)表達(dá)分析
組織：直徑0.2 m，0.2 g總蛋白，只是傳統(tǒng)western blot的幾十分之一；
原代神經(jīng)元細(xì)胞：1 g總蛋白
如原文方法學(xué)描述
Protein analysis using Wes
10-weeks-old DAT-iCre line 6 transgenic and WT rats (7 rats from each line) unilaterally injected with AAV Cas9 and AAV LSL-Th gRNA into the midbrain were analyzed for TH protein expression through capillary western blot (Wes, Protein simple, San Jose, CA). Six weeks after injection, the animals were anesthetized and the brains were removed. The left and right midbrain regions were collected by taking a 2 m diameter punch of the tissue. Tissues were lysed in a modified RIPA buffer (described above), sonicated 10 s at 10 A (Ultrasonic Processor GE 70, Cole Parmer, Vernon Hills, IL), and then centrifuged at 11,000 rpm for 15 min at 4_C.After quantification of protein concentration in the supernatant using a DC assay (Bio-Rad), 0.2 g of total protein was transferred to 12-230 kDa Separation Modules (Protein Simple) for separation and detection using mouse anti-TH (1:50; Millipore #MAB318, RRID: AB_2201528) and mouse anti-actin (1:50; Cell Signal Technology mAb 3700S, RRID: AB_2242334) primary antibodies, and the Anti-Mouse Detection Module (Protein Simple,). The relative amount of TH protein was determined using the areas under peaks from the chemiluminescence chromatograms provided by the Compass for SW software (version 4.0.0, Protein Simple). TH values were normalized to actin before relating the injected left side to the non-injected right side.
For protein detection in primary cortical neuron cultures, cells were lysed in RIPA buffer (above) and 1 g protein was loaded onto WES 25-well plates for separation of 12-230 kDa proteins (ProteinSimple). MANF and actin proteins were detected using rabbit anti-MANF (YenZym) and mouse anti-actin (Abcam #ab3280) as primary antibodies, and HRP-conjugated anti-rabbit and anti-mouse secondary antibodies according to the instructions in the WES separation kit (ProteinSimple).
中腦多巴胺能神經(jīng)元與帕金森、藥物濫用密切相關(guān)
帕金森病和藥物濫用障礙都與中腦多巴胺能神經(jīng)元的功能改變有關(guān)。在外側(cè)中腦，黑質(zhì)(SN)的多巴胺能神經(jīng)元(SN)向背側(cè)紋狀體發(fā)出投射，參與控制運(yùn)動(dòng)，而位于腹側(cè)被蓋區(qū)(VTA)的多巴胺能神經(jīng)元(VTA)支配腹側(cè)紋狀體(伏隔核)和前額葉皮質(zhì)，以參與激勵(lì)和獎(jiǎng)勵(lì)方式。因此，在疾病進(jìn)展的不同階段改變中腦多巴胺能神經(jīng)元的遺傳組成的能力，可以揭示基因在相關(guān)病理表現(xiàn)中的作用，從而促進(jìn)新的治療方法的發(fā)展。
特定基因敲除建立中腦多巴胺能神經(jīng)元研究模型
美國(guó)NIH下屬美國(guó)國(guó)家藥物濫用研究中心（National Institute on Drug Abuse）的多個(gè)研究所，聯(lián)合在神經(jīng)生物學(xué)頂級(jí)雜志Neuron發(fā)表研究結(jié)果：Neuron-Specific Genome Modification in the Adult Rat Brain Using CRISPR-Cas9 Transgenic Rats，Neuron (2019), doi.org/10.1016/j.neuron.2019.01.035 通過(guò)CRISPR-Cas9技術(shù)建立特定神經(jīng)元敲除大鼠模型。
研究摘要
從歷史上看，大鼠一直是行為研究的首選動(dòng)物模型。然而，基因組修飾方面的限制造成了與現(xiàn)有轉(zhuǎn)基因小鼠相比，它們的使用滯后。本文，科學(xué)家們開(kāi)發(fā)了幾種轉(zhuǎn)基因工具，包括病毒載體和轉(zhuǎn)基因大鼠，利用CRISPR-Cas9技術(shù)對(duì)特定成年大鼠神經(jīng)元進(jìn)行靶向基因組修飾。從野生型大鼠開(kāi)始，利用腺相關(guān)病毒(AAV)表達(dá)Cas9或?qū)騌NA(GRNAs)的載體，實(shí)現(xiàn)酪氨酸羥化酶的敲除。隨后構(gòu)建了一個(gè)AAV載體，用于Cre依賴(lài)的gRNA表達(dá)，以及三個(gè)新的轉(zhuǎn)基因大鼠系，將CRISPR-Cas9組分靶向到多巴胺能神經(jīng)元。一只大鼠代表第一只以精原干細(xì)胞為靶點(diǎn)的生殖細(xì)胞基因敲除大鼠模型。本文所描述的大鼠是一種通用的平臺(tái)，用于在成年大鼠的大腦和其他可能表達(dá)cre的組織中進(jìn)行細(xì)胞特異性和序列特異性基因組修飾。
Wes在本文中被用于微量中腦組織和原代神經(jīng)元細(xì)胞蛋白質(zhì)表達(dá)分析
組織：直徑0.2 m，0.2 g總蛋白，只是傳統(tǒng)western blot的幾十分之一；
原代神經(jīng)元細(xì)胞：1 g總蛋白
如原文方法學(xué)描述
Protein analysis using Wes
10-weeks-old DAT-iCre line 6 transgenic and WT rats (7 rats from each line) unilaterally injected with AAV Cas9 and AAV LSL-Th gRNA into the midbrain were analyzed for TH protein expression through capillary western blot (Wes, Protein simple, San Jose, CA). Six weeks after injection, the animals were anesthetized and the brains were removed. The left and right midbrain regions were collected by taking a 2 m diameter punch of the tissue. Tissues were lysed in a modified RIPA buffer (described above), sonicated 10 s at 10 A (Ultrasonic Processor GE 70, Cole Parmer, Vernon Hills, IL), and then centrifuged at 11,000 rpm for 15 min at 4_C.After quantification of protein concentration in the supernatant using a DC assay (Bio-Rad), 0.2 g of total protein was transferred to 12-230 kDa Separation Modules (Protein Simple) for separation and detection using mouse anti-TH (1:50; Millipore #MAB318, RRID: AB_2201528) and mouse anti-actin (1:50; Cell Signal Technology mAb 3700S, RRID: AB_2242334) primary antibodies, and the Anti-Mouse Detection Module (Protein Simple,). The relative amount of TH protein was determined using the areas under peaks from the chemiluminescence chromatograms provided by the Compass for SW software (version 4.0.0, Protein Simple). TH values were normalized to actin before relating the injected left side to the non-injected right side.
For protein detection in primary cortical neuron cultures, cells were lysed in RIPA buffer (above) and 1 g protein was loaded onto WES 25-well plates for separation of 12-230 kDa proteins (ProteinSimple). MANF and actin proteins were detected using rabbit anti-MANF (YenZym) and mouse anti-actin (Abcam #ab3280) as primary antibodies, and HRP-conjugated anti-rabbit and anti-mouse secondary antibodies according to the instructions in the WES separation kit (ProteinSimple).
