分泌性胚胎碱性磷酸酶的测定

Meiyan Wang; Xinyi Wang; Haifeng Ye

doi:10.21769/BioProtoc.4600

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Peer-reviewed

Measurement of Secreted Embryonic Alkaline Phosphatase

分泌性胚胎碱性磷酸酶的测定

MW Meiyan Wang

XW Xinyi Wang

HY Haifeng Ye email

发布: 2023年02月05日第13卷第3期 DOI: 10.21769/BioProtoc.4600 浏览次数: 1882

评审: Laxmi Narayan MishraNityanand SrivastavaSashikantha Reddy Pulikallu

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Abstract

Secreted reporters have been demonstrated to be simple and useful tools for analyzing transcriptional regulation in mammalian cells. The distinctive feature of these assays is the ability to detect reporter gene expression in the culture supernatant without affecting the cell physiology or leading to cell lysis, which allows repeated experimentation and sampling of the culture medium using the same cell cultures. Secreted embryonic alkaline phosphatase (SEAP) is one of the most widely used reporter, which can be easily detected using colorimetry following incubation with a substrate, such as p-nitrophenol phosphate. In this report, we present detailed procedures for detection and quantification of the SEAP reporter. We believe that this step-by-step protocol can be easily used by researchers to monitor and measure molecular genetic events in a variety of mammalian cells due to its simplicity and ease of handling.

Graphical abstract

Schematic overview of the workflow described in this protocol

Keywords: SEAP (分泌性胚胎碱性磷酸酶)

Secreted reporter (分泌性报告)

Reporter gene assays (报告基因分析)

Gene expression (基因表达)

Gene regulation (基因调控)

Background

Reporter gene assays have been important for biomedical and pharmaceutical studies to monitor cellular activities associated with gene expression, regulation, and signal transduction. An ideal secreted reporter system would provide a more simple, rapid, and sensitive method to detect gene expression in a quantitative manner compared to classical intracellular reporters. Secreted embryonic alkaline phosphatase (SEAP), a C-terminal truncated variant of human placental alkaline phosphatase (PLAP), is one of the most widely used secreted mammalian reporter enzymes, engineered by deleting the 30 amino acid anchoring domain of PLAP (Berger et al., 1988). The SEAP reporter assay has superior properties: it is a simple and non-invasive procedure that does not require cell lysis and allows for long-term monitoring of gene expression in vitro and in vivo (Yang et al., 1997; Schlatter et al., 2002; Jiang et al., 2008). Moreover, transfected cells are not disturbed and remain intact for further studies during measurement of SEAP activity (Hu et al., 2022). Furthermore, the heat stability of SEAP and resistance to the phosphatase inhibitor L-homoarginine allow the samples to be pretreated at 65°C or with this inhibitor, to eliminate endogenous alkaline phosphatase activity (Tannous and Teng, 2011). Here, we describe the experimental procedures of in vitro measurement of SEAP adapted from our previously published work (Wang et al., 2021). This method can be useful for real-time monitoring of gene expression patterns in various cells and animals.

Materials and Reagents

96-well plate, transparent (Corning, catalog number: 3365)
24-well plate, transparent (Corning, catalog number: 3537)
T25 flask (Corning, catalog number: 430639)
HEK-293T (ATCC, CRL-11268)
Dulbecco's modified Eagle's medium (DMEM) (Gibco, catalog number: 31600-083)
Fetal bovine serum (FBS) (Gibco, catalog number: 10270-106)
0.25% trypsin-EDTA
1% (vol/vol) penicillin and streptomycin solution (Beyotime Inc., catalog number: ST488-1/ST488-2)
p-nitrophenylphosphate (Sangon Biotech, CAS: 333338-18-4)
L-homoarginine (Sangon Biotech, CAS: 1483-01-8)
MgCl₂·6H₂O (Sangon Biotech, CAS: 7791-18-6)
Diethanolamine (Sangon Biotech, CAS: 111-42-2)
NaCl (Sangon Biotech, CAS: 7647-14-5)
KCl (Sangon Biotech, CAS: 7647-40-7)
Na₂HPO₄ (Sangon Biotech, CAS: 7558-79-4)
KH₂PO₄ (Sangon Biotech, CAS: 7778-77-0)
HCl (Sinopharm Chemical Reagent Co., Ltd., CAS: 7647-01-0）
Polyethyleneimine (PEI, molecular weight 40,000, stock solution 1 mg/mL in ddH₂O) (Polysciences, catalog number: 24765)
1× PBS (see Recipes)
2× SEAP assay buffer (see Recipes)
Substrate solution (see Recipes)

Plasmids (see Table 1)

Table 1.Plasmids designed and used in this study

Plasmid	Description and cloning strategy	Reference
pXY137	Constitutive mammalian BldD and BphS expression vector (ITR-P_hCMV-p65-VP64-BldD-pA-PhCMV-BphS-P2A-YhjH-pA-ITR)	Shao et al. (2017)
pZQ5	Far-red light-inducible expression vector (2*crRNA binding site-P_hCMVmin-SEAP-pA)	This work
pZQ28	Constitutive crRNA2_SEAP expression vector (P_U6-crRNA_SEAP-pA)	This work
pZQ113	Constitutive mammalian P_hCMV-driven expression vector (P_hCMV-scFv-45bp linker-p65-HSF1-pA)	This work
pZQ116	Far-red light-inducible expression vector (P_FRL2-dAsCas12a-NLS-10×GCN4-pA; P_FRL3, (OwhiG)₂-P_hCMVmin)	This work

Equipment

Water bath
CO₂ incubator
Constant temperature incubator (65°C)
Synergy H1 hybrid multi-mode microplate reader (BioTek Instruments, Inc.)
Multipass pipette
Reagent reservoirs
Hemocytometer (Merck, catalog number: Z359629)

Software

Excel (Microsoft)
Gen5 software (version: 2.04)

Procedure

English

中文翻译

文章信息

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如何引用

Readers should cite both the Bio-protocol article and the original research article where this protocol was used:

Wang, M., Wang, X. and Ye, H. (2023). Measurement of Secreted Embryonic Alkaline Phosphatase. Bio-protocol 13(3): e4600. DOI: 10.21769/BioProtoc.4600.
Wang, X., Dong, K., Kong, D., Zhou, Y., Yin, J., Cai, F., Wang, M. and Ye, H. (2021). A far-red light-inducible CRISPR-Cas12a platform for remote-controlled genome editing and gene activation. Science advances 7(50): eabh2358.