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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

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 method3,4. Studies have shown that pigs can be used as a potential source of human organs for clinical transplantation5,6. 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-.......

Representative 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.......

Acknowledgements

This work were supported by grants from National Key R&D Program of China (2017YFC1103704), Shenzhen Foundation of Science and Technology (JCYJ20170817172116272), Special Funds for the Construction of High Level Hospitals in Guangdong Province (2019), Sanming Project of Medicine in Shenzhen (SZSM201412020), Fund for High Level Medical Discipline Construction of Shenzhen (2016031638), Shenzhen Foundation of Health and Family Planning Commission (SZXJ2017021, SZXJ2018059).

....

Materials

NameCompanyCatalog NumberComments
AgaroseBiowest, Barcelona, Spain111860
BamHI-HFNew England Biolabs, Ipswich, Mass, USAR3136S
1.5 mL microcentrifuge tubeAxygen Biosciences, Union City, CA, USAMCT-150-C
0.2 mL PCR tubeAxygen Biosciences, Union City, CA, USAPCR-02-C
C57BL/6 MiceMedical Animal Center of Guangdong Province,  China8~10 weeks
CentrifugeThermo Fisher Scientific, Walt- ham, MA, USAMicro 21R
2-Log DNA LadderNew England Biolabs, Ipswich, Mass, USAN3200S0.1–10.0 kb
Marker I Tiangen, Beijing, ChinaMD101-020.1–0.6 kb
DNA Mini Column(HiBind DNA Mini Columns)Omega Bio-tek, Norcross, GA, USADNACOL-01
DNA loading bufferSolarbio, Beijing, ChinaD1010
E.Z.N.A.Plasmid DNA Mini Kit I and E.Z.N.A. Plasmid DNA Mini Kit IIOmega Bio-tek, Norcross, GA, USAD6942
D6943
EcoR ITakara Bio, Shiga, Japan 1040S
Female Bama mini pigsBGI Ark Biotechnology, Shenzhen, China2~4 months
Genomic DNA Extraction Kit ⅠTiangen, Beijing, ChinaDP304-02
SYBR Green Realtime PCR Master MixToyobo, Osaka, JapanQPK-201
Gel Doc XRBio-Rad, Hercules, USA
Male cynomolgus monkeysGuangdong Landau Biotechnology, Guangzhou, China8 years
Nucleic acid dye(Gelred)Biotium, Fremont, USA42003
polymerase(Premix Taq)Takara Bio, Shiga, JapanRR901A
pMD19-T plasmidTakara Bio, Shiga, JapanD102A
qPCR machineApplied Biosystems QSDx, Waltham, USA
Serum/Circulating DNA Extraction KitTiangen, Beijing, ChinaDP339
TAEsangon Biotech, Shanghai, ChinaB548101

References

  1. Buchman, T. Multiple organ failure. Current Opinion In General Surgery. , 26-31 (1993).
  2. Smith, M., Dominguez-Gil, B., Greer, D., Manara, A., Souter, M. Organ donation after circulatory death: current status and future potential.

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XenotransplantationPig specific DNABloodMonitoringImmune RejectionPCRDNA ExtractionAgarose Gel Electrophoresis

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