Quantification of Circulating Pig-Specific DNA in the Blood of a Xenotransplantation Model

Summary

In this protocol, porcine specific primers were designed, plasmids-containing porcine specific DNA fragments were constructed, and standard curves for quantitation were established. Using species-specific primers, cpsDNA was quantified by qPCR in pig-to-mouse cell transplantation models and pig-to-monkey artery patch transplantation models.

Abstract

Xenotransplantation is a feasible method to treat organ failure. However, how to effectively monitor the immune rejection of xenotransplantation is a problem for physicians and researchers. This manuscript describes a simple and effective method to monitor immune rejection in pig-to-mouse cell transplantation models and pig-to-monkey artery patch transplantation models. Circulating DNA is a potentially non-invasive biomarker for organ damage. In this study, circulating pig-specific DNA (cpsDNA) was monitored during xenograft rejection by quantitative real-time PCR (qPCR). In this protocol, porcine specific primers were designed, plasmids-containing porcine specific DNA fragments were constructed, and standard curves for quantitation were established. Species-specific primers were then used to quantify cpsDNA by qPCR in pig-to-mouse cell transplantation models and pig-to-monkey artery patch transplantation models. The value of this method suggests that it can be used as a simple, convenient, low cost, and less invasive method to monitor the immune rejection of xenotransplantation.

Introduction

Organ failure is one of the main causes of death¹. Transplantation of cells, tissues and organs is an effective way to treat organ failure². Nevertheless, the shortage of donor organs limits the clinical application of this method^3,4. Studies have shown that pigs can be used as a potential source of human organs for clinical transplantation^5,6. However, cross-species organ transplantation faces dangerous immune rejection. Therefore, it is crucial to monitor the immune rejection of xenotransplantation. Cur....

Protocol

All experiments were performed in accordance with the relevant guidelines and regulations of the Institutional Review Board of Shenzhen Second People's Hospital, First Affiliated Hospital of Shenzhen University.

1. Design porcine specific primers

1. Perform whole-genome BLAST analysis to identify porcine specific genes that were different from those of humans, monkeys or mouse, using the software NCBI (www.ncbi.nlm.nih.gov).
2. Design primers according to 19 pig-specific genes (Table 1) using the software of Primer 5.

2. Isolating genomic DNA

Results

In this protocol, porcine specific primers were designed, plasmids-containing porcine specific DNA fragments were constructed, and standard curves for quantitation were established (Figure 4). Species specificities of the 19 primers were confirmed by PCR. Species-specific primers (primer #4 and primer #11) were then used to quantify cpsDNA by qPCR in pig-to-mouse cell transplantation models and pig-to-monkey artery patch transplantation models.

Agarose electrophor.......

Discussion

Quantifying porcine circulating DNA represents a feasible approach to monitor the immune rejection of xenotransplantation. Gadi et al.²⁴ found that donor-derived circulating DNA (ddcfDNA) content in the blood of patients with acute rejection was significantly higher than that of patients without rejection. These studies suggest that ddcfDNA may be a common biomarker for monitoring organ graft damage. In recent years, qPCR has increasingly been applied to the analysis of nucleic acids because of it.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Agarose	Biowest, Barcelona, Spain	111860
BamHI-HF	New England Biolabs, Ipswich, Mass, USA	R3136S
1.5 mL microcentrifuge tube	Axygen Biosciences, Union City, CA, USA	MCT-150-C
0.2 mL PCR tube	Axygen Biosciences, Union City, CA, USA	PCR-02-C
C57BL/6 Mice	Medical Animal Center of Guangdong Province,  China		8~10 weeks
Centrifuge	Thermo Fisher Scientific, Walt- ham, MA, USA	Micro 21R
2-Log DNA Ladder	New England Biolabs, Ipswich, Mass, USA	N3200S	0.1–10.0 kb
Marker I	Tiangen, Beijing, China	MD101-02	0.1–0.6 kb
DNA Mini Column(HiBind DNA Mini Columns)	Omega Bio-tek, Norcross, GA, USA	DNACOL-01
DNA loading buffer	Solarbio, Beijing, China	D1010
E.Z.N.A.Plasmid DNA Mini Kit I and E.Z.N.A. Plasmid DNA Mini Kit II	Omega Bio-tek, Norcross, GA, USA	D6942
D6943
EcoR I	Takara Bio, Shiga, Japan	1040S
Female Bama mini pigs	BGI Ark Biotechnology, Shenzhen, China		2~4 months
Genomic DNA Extraction Kit ?	Tiangen, Beijing, China	DP304-02
SYBR Green Realtime PCR Master Mix	Toyobo, Osaka, Japan	QPK-201
Gel Doc XR	Bio-Rad, Hercules, USA
Male cynomolgus monkeys	Guangdong Landau Biotechnology, Guangzhou, China		8 years
Nucleic acid dye(Gelred)	Biotium, Fremont, USA	42003
polymerase(Premix Taq)	Takara Bio, Shiga, Japan	RR901A
pMD19-T plasmid	Takara Bio, Shiga, Japan	D102A
qPCR machine	Applied Biosystems QSDx, Waltham, USA
Serum/Circulating DNA Extraction Kit	Tiangen, Beijing, China	DP339
TAE	sangon Biotech, Shanghai, China	B548101