Construction of POR gene knockout, complementation and overexpression LO2 cell lines and preliminary application as AFB1 exposed model

doi:10.3969/j.issn.1004-1524.20230325

Acta Agriculturae Zhejiangensis ›› 2024, Vol. 36 ›› Issue (2): 272-283.DOI: 10.3969/j.issn.1004-1524.20230325

• Animal Science • Previous Articles Next Articles

Construction of POR gene knockout, complementation and overexpression LO2 cell lines and preliminary application as AFB₁ exposed model

WANG Lin¹^,²(), YUAN Jianlin¹^,², MIAO Chang¹^,², MA Yuhan¹^,², CAO Sanjie¹^,², ZHAO Qin¹^,²^,^*()

1. Research Center for Swine Diseases, College of Veterinary Medicine, Sichuan Agricultural University, Chengdu 611130, China
2. Sichuan Science-Observation Experimental Station of Veterinary Drugs and Veterinary Diagnostic Technique, Ministry of Agriculture and Rural Affairs, Chengdu 611130, China

Received:2023-03-14 Online:2024-02-25 Published:2024-03-05

Abstract

Abstract:

To construct three LO2 cell lines separately with POR gene stable knockout (POR^KO), complementation (POR^CO), and overexpression (POR^OE), and provide cell models for further in-depth research on the function of the POR gene in the mechanism of AFB₁toxicity. Firstly, two sgRNAs targeting the POR gene were designed using CRISPR/Cas9 technology and cloned into the lentiCRISPR v2 vector, and then lentivirus packaging and virus infection were performed. After screening LO2 POR^KO monoclonal cells, they were sequenced and identified by Western blot. Secondly, the pcDNA3.1(+)/myc-His B-POR recombinant plasmid was constructed by homologous recombination method, and the LO2 POR^CO and LO2 POR^OE cell lines were respectively constructed through cell transfection and were identified by Western blot after screening with G-418. After the wild-type LO2, LO2 POR^KO, LO2 POR^CO, and LO2 POR^OE cell lines were exposed with 4 μmol·L^-1 and 8 μmol·L^-1 AFB₁for 48 h, then the cell morphology was observed, and the cell viability was determined. The results showed that sgRNA-2 could successfully knock out the POR gene to obtain the LO2 POR^KO cell line, and the LO2 POR^CO and LO2 POR^OE cell lines were successfully constructed. After each cell line was exposed to 4 μmol·L^-1 and 8 μmol·L^-1 AFB₁for 48 h, compared with the wild-type LO2 cell viability rate of 43.66%±0.58% and 27.90%±0.46%, the cell viability rate of LO2 POR^KO cells was extremely significant, both 100% (P<0.000 1); the cell viability rate of LO2 POR^CO cells was still significantly different, respectively 57.07%±0.74% and 42.54%±0.68% (P<0.05); the cell viability rate of LO2 POR^OE cells was significantly lower, respectively 24.58%±0.92% and 17.99%±0.81% (P<0.01). In conclusion, this paper successfully constructed LO2 POR^KO, LO2 POR^CO, and LO2 POR^OE cell lines, and studied the cell viability rate and cell morphology changes of normal LO2, LO2 POR^KO, LO2 POR^CO, and LO2 POR^OE cell lines with AFB₁exposure, and discovered that the POR gene has a huge impact on AFB₁-induced cytotoxicity. This paper laid a foundation for the subsequent in-depth research on the function of the POR gene in the mechanism of AFB₁toxicity.

Key words: POR, CRISPR/Cas9, LO2 cells, AFB₁

CLC Number:

S851.31

WANG Lin, YUAN Jianlin, MIAO Chang, MA Yuhan, CAO Sanjie, ZHAO Qin. Construction of POR gene knockout, complementation and overexpression LO2 cell lines and preliminary application as AFB₁ exposed model[J]. Acta Agriculturae Zhejiangensis, 2024, 36(2): 272-283.

Figures/Tables 11

Table 1 sgRNA and corresponding primer sequences

靶位点 Target	靶位点序列 Target sequence (5'→3')	引物序列 Primer sequence(5'→3')
sgRNA-POR-1	AGAGATCGACAACGCCCTGG	F: ACCGAGAGATCGACAACGCCCTGG R: AAACCCAGGGCGTTGTCGATCTCTC
sgRNA-POR-2	GCCAGAGATCGACAACGCCC	F:GACCGCCAGAGATCGACAACGCCC R:AAACGGGCGTTGTCGATCTCTGGC

Table 2 POR gene deletion identification primer sequence

靶位点 Target	敲除鉴定引物序列 Primer sequence(5'→3')
sgRNA-POR-1	hPOR-KO1-F: AACCTTGAACAGGCTCAGTCAT hPOR-KO1-R: ATACATGGATCTGGAGTCCCC
sgRNA-POR-2	hPOR-KO2-F: AGCCCCTCCGTGTTGTTA hPOR-KO2-R: AGGGGACATTCTCGTAGTGC

Table 3 POR homologous amplified primer sequence

引物名称 Primer name	引物序列 Primer sequence(5'→3')
POR-F	ctaccggactcagatctcgagATGATCAACATGGGAGACTCCC
POR-R	gtaccgtcgactgcagaattcCTAGCTCCACACGTCCAGGG

Fig.1 Construction of lentiCRISPR v2-sgRNA-POR recombinant plasmid A, lentiCRISPR v2 plasmid information partial schematic diagram and sgRNA insertion location; B, sgRNA sequence and target site diagram; C, lentiCRISPR v2-sgRNA recombinant plasmid sequencing results.

Fig.2 Identification of LO2 PORKO monoclonal cell lines A, LO2 PORKO cell sequencing peak map; B, LO2 PORKO cell sequence comparison map.

Fig.3 Construction of pcDNA3.1(+)/myc-His B-POR recombinant eukaryotic expression plasmid A, pcDNA3.1(+)/myc-His B plasmid partial information schematica and insertion site of POR target fragment; B, POR homologous fragment amplified in LO2 cell cDNA as template,1 is DNA marker,2 is the target fragment; C, PCR electrophoretic map of colony identification, 1 is DNA marker, 2-4 is positive colony; D, Sequencing results of positive colonies.

Fig.4 Results of WB identification of cell lines With LO2 cells as the control, WB detected the expression of POR protein in LO2 PORKO, LO2 PORCO and LO2 POROE cells, and the amino acid sequences in LO2 PORKO cells were analyzed.

Fig.5 The toxic dose-effect curve of AFB1on the relative cell viability of wild-type LO2 cell lines

Table 4 The results of IC values

毒素IC值 Toxin IC value		浓度的95%置信限度 95% confidence limit for concentration/(μmol·L^-1)			毒素IC值 Toxin IC value		浓度的95%置信限度 95% confidence limit for concentration/(μmol·L^-1)
毒素IC值 Toxin IC value		估算 Estimated value	上限 Upper limit	下限 Lower limit	毒素IC值 Toxin IC value		估算 Estimated value		上限 Upper limit		下限 Lower limit
AFB₁	IC₁₀	1.57	0.00	4.44	AFB₁	IC₁₀	0.22	0.02		0.65
24 h	IC₁₅	4.97	0.10	9.37	48 h	IC₁₅	0.41	0.05		1.03
	IC₂₀	11.43	3.34	25.27		IC₂₀	0.66	0.12		1.46
	IC₂₅	23.11	12.68	270.59		IC₂₅	0.97	0.22		1.94
	IC₃₀	42.74	21.06	4.14×10³		IC₃₀	1.36	0.37		2.50
	IC₃₅	74.72	30.47	5.41×10⁴		IC₃₅	1.86	0.61		3.15
	IC₄₀	126.03	42.02	6.14×10⁵		IC₄₀	2.48	0.96		3.92
	IC₄₅	208.04	56.62	6.37×10⁶		IC₄₅	3.27	1.49		4.86
	IC₅₀	339.97	75.45	6.33×10⁷		IC₅₀	4.28	2.26		6.05
	IC₆₀	917.09	133.59	6.62×10⁹		IC₆₀	7.41	5.02		9.89
	IC₇₀	2 704.23	247.31	1.06×10¹²		IC₇₀	13.45	10.07		20.06
	IC₈₀	1.01×10⁴	521.74	5.18×10¹⁴		IC₈₀	27.86	18.96		59.05
	IC₉₀	7.36×10⁴	1.60×10³	5.80×10¹⁸		IC₉₀	83.30	43.69		336.79
	IC₉₉	2.60×10⁴	4.30×10⁴	5.52×10³⁰		IC₉₉	2.12×10³	465.01		6.44×10⁴

Fig.6 Effect of morphological characteristics on cells after 48 h treated with AFB1under optical microsope (100×)

Fig.7 Effect of AFB1treatment on cell viability The cell viability of LO2, LO2 PORKO, LO2 PORCO and POROE cells treated with AFB1for 48 hours. Different cell lines at different concentrations were compared with negative control groups, **, P<0.01; ***, P<0.001; ns, P>0.05. Compared with LO2 cell group under the same concentration of AFB1, *, P<0.05; **, P<0.01; ****, P<0.000 1.

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Construction of POR gene knockout, complementation and overexpression LO2 cell lines and preliminary application as AFB₁ exposed model

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