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. Currently, clinical monitoring of immune rejection depends mainly on the patient's signs and symptoms, as well as laboratory tests (e.g., biopsy, immunobiochemical analysis, and ultrasound)^7,8,9. However, these monitoring methods have many disadvantages. The signs and symptoms of immune rejection in patients usually appear late¹⁰, which is not conducive to early detection and early intervention; biopsy has the disadvantage of being invasive¹¹, which is not easy for patients to accept; immunobiochemical analysis lacks sensitivity or specificity, and ultrasound is auxiliary and expensive. Therefore, it is urgent to find an effective and convenient method to monitor the immune rejection.

Circulating DNA is an extracellular type of DNA found in blood. Mandel and Metais¹² first reported the presence of circulating DNA in peripheral blood in 1948. Under normal physiological conditions, circulating DNA in the blood of healthy people is relatively low at baseline. However, in some pathologies, such as tumors, myocardial infarction, autoimmune diseases, and transplant rejection, the level of circulating DNA in the blood can be significantly increased^13,14 due to the massive release of circulating DNA caused by apoptosis and necrosis. The origin of circulating DNA is associated with apoptosis and necrosis¹⁵, which are characteristic of xenograft rejection¹⁶.

Circulating DNA has been proven to be a minimally-invasive biomarker for detecting cancers^17,18,19. High through-put sequencing of donor-derived circulating DNA is reliable for the detection of rejection after organ transplantation^20,21. However, this method requires a high concentration and quality of extracted DNA. The DNA requirements in addition to the high cost and time-consumption make this method ineligible for routine clinical use. Donor-derived circulating DNA can be precisely quantified by quantitative real-time PCR (qPCR), which is both specific and sensitive. Therefore, quantifying porcine circulating DNA by qPCR is a feasible method to monitor the immune rejection of xenotransplantation. This is less invasive, highly sensitive and specific, low cost, and time-saving. Pigs and human beings are genetically separate with quite different genomic sequences (Figure 1). Therefore, circulating porcine DNA can be released into the recipient's blood post-xenotransplantation because of xeno-rejection. CpsDNA could be precisely quantified by qPCR with species-specific primers in the recipient’s blood. Previously, we have demonstrated the rationale and feasibility of cpsDNA as a biomarker for xenotransplantation^22,23. Here, we disclose more experimental tips and details. The experiment consists of the following steps. Firstly, porcine specific primers were designed, and genomic DNA was isolated, which were used to verify the specificity of the primers by regular PCR. Secondly, constructing the standard curve of cpsDNA and isolating cpsDNA from the sample blood. Finally, the circulating pig-specific DNA was quantified using qPCR.

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

NOTE: The genomic DNA (including blood from pigs, monkeys, volunteer humans, monkeys with pig grafts, and mice with porcine cells) were extracted using a commercial genomic DNA extraction kit (Table of Materials).

1. Place 500 µL of the whole blood of above samples into different microcentrifuge tubes, respectively.
2. Add 20 µL of protease K and 500 µL of lysis buffer into the above microcentrifuge tubes, and then thoroughly shake and mix.
3. Put these microcentrifuge tubes in a water bath at 56 °C for 10 min and shake 2-3 times during this process until the solution becomes clear.
4. Centrifuge briefly to remove the liquid beads from the inner wall of the tube covers. Add 500 µL of anhydrous ethyl alcohol and shake thoroughly.
5. Transfer the mixture into the adsorption column, centrifuge at 12,000 rpm (~13,400 x g) for 2 min.
6. Add 800 µL of rinse solution to each adsorption column; centrifuge for 1 min at 12,000 rpm ( ~13,400 x g). Place the columns at room temperature for a few minutes to dry the remaining rinse solution.
   NOTE: The rinse solution is provided by the manufacturer.
7. Transfer the adsorption columns to another clean centrifugal tube, add 50 µL of elution buffer to the middle of the adsorption films, and place them at room temperature for 2-5 min. Centrifuge 6,200 x g for 1 min 12,000 rpm (~13,400 x g).
   NOTE: The elution buffer is provided by the manufacturer, but the TE buffer, containing 10 mM Tris-HCl (pH 8.0) and 0.1 mM EDTA, can also elute the DNA.
8. Store the DNA solution at -20 °C.

3. Verify the specificity of the primers

NOTE: Species specificities of the above 19 primers were confirmed by PCR, which was performed using polymerase (Table of Materials) and primers presented in Table 1.

1. Prepare the pre-mixed solution of 12.5 µL of 2x PCR premix (Table of Materials), 1 µL of 5' primer (10 µM), 1 µL of 3' primer (10 µM), and 8.5 µL of ddH₂O. Prepare the mix including 2 extra samples.
2. Split 23 µL of pre-mixed solution into 0.2 mL microcentrifuge tubes, add 2 µL of genomic DNA, and carefully cap the tubes. Then mix and centrifuge slightly.
3. Place the 0.2 mL microcentrifuge tubes into the PCR-cycler and perform the following: Denaturation: 95 °C for 5 s; Annealing: 60 °C for 30 s; extension:72 °C for 30 s.
4. Perform agarose electrophoresis as follows:
   1. Weigh 1.2 g of agarose into a flask containing 100 mL of 1x TAE and boil it for 5 min in the microwave. Add 5 µL of nucleic acid dye (Table of Materials) into the flask after it cools to about 70 °C. Pour it into the plate along the edge slowly, and place it at room temperature until it solidifies into a gel.
   2. Add 5 µL of sample and 2-Log DNA Ladder (0.1–10.0 kb) or Marker I (0.1–0.6 kb), which contain 1 µL of 6x DNA loading buffer, into the agarose gel. Then electrophorese at 120 mA until the bands are separated.
   3. Visualize the agar gel containing DNA fragments with an ultraviolet imager.
5. Perform agarose electrophoresis to isolate amplified DNA fragments from the above samples, which was visualized in an ultraviolet imager. Using the PCR, the primers specific for amplified porcine genomic DNA was first identified (Figure 2A). Further, certain species-specificities of these primers which specifically amplified pig DNA in the cohort of monkey/human genomic or in the cohort of mouse genomic DNA were confirmed (Figure 2B). Finally, two species-specificities primers were further proven to specifically amplify pig DNA in the pig-to-monkey artery patch and/or pig-to-mouse cell transplantation models, respectively (Figure 2C)

4. Standard curve of cpsDNA

1. Transform the pMD19-T plasmid containing a fragment of porcine DNA into DH5a, which could be specifically amplified by primer #4 or primer #11 (Table 1). Screen for positive bacteria using 50 µg/mL ampicillin.
   Primer #4 in human/monkey cohort (forward: 5′-TTCAATCCCA CTTCTTCCACCTAA-3′, reverse: 5′-CTTCATTCCATCTTCATAATAAC CCTGT-3′)
   Primer #11 for mouse model (forward: 5′-TGCCGTGGTTTCC GTTGCTTG-3′, reverse: 5′-TCACATTTGATGGTCGTCTTGTCGTC T-3′)
   NOTE: Details of all the primers can be found in Table 1.
2. Harvest the above plasmids following the protocol below.
   1. Isolate a single colony from a freshly streaked selective plate and inoculate a culture of 1- 5 mL of LB medium containing the appropriate selective antibiotic. Incubate for 12-16 hours at 37 °C with vigorous shaking (300 rpm).
   2. Centrifuge at 10,000 x g for 1 minute at room temperature. Decant or aspirate and discard the culture media.
   3. Add 250 µL of Solution I/RNase A. Vortex or pipet up and down to mix thoroughly. Complete resuspension of cell pellet is vital for obtaining good yields.
      NOTE: RNase A must be added to Solution I before use.
   4. Transfer suspension into a new 1.5 mL microcentrifuge tube. Add 250 µL of Solution II. Invert and gently rotate the tube several times to obtain a clear lysate. A 2-3 minutes incubation may be necessary.
      NOTE: Avoid vigorous mixing as this will shear chromosomal DNA and lower plasmid purity. Do not allow the lysis reaction to proceed more than 5 minutes.
   5. Add 350 µL of Solution III. Immediately invert several times until a flocculent white precipitate forms.
      NOTE: It is vital that the solution is mixed thoroughly and immediately after the addition of Solution III to avoid localized precipitation.
   6. Centrifuge at maximum speed (≥13,000 x g) for 10 minutes. A compact white pellet will form. Promptly proceed to the next step.
   7. Insert a DNA mini column into a 2 mL collection tube.
   8. Transfer the cleared supernatant from 4.2.7 by carefully aspirating it into the DNA mini column. Be careful not to disturb the pellet and that no cellular debris is transferred to the DNA mini column.
   9. Centrifuge at maximum speed for 1 minute. Discard the filtrate and reuse the collection tube.
   10. Add 500 µL of HBC Buffer. Centrifuge at maximum speed for 1 minute. Discard the filtrate and reuse collection tube.
       NOTE: HBC Buffer must be diluted with 100% isopropanol before use.
   11. Add 700 µL of DNA Wash Buffer. Centrifuge at maximum speed for 1 minute. Discard the filtrate and reuse the collection tube.
       NOTE: DNA Wash Buffer must be diluted with 100% ethanol prior to use.
       1. Optional: Repeat for a second DNA wash buffer wash step.
   12. Centrifuge the empty DNA mini column for 2 minutes at maximum speed to dry the column matrix.
       NOTE: It is important to dry the DNA mini column matrix before elution. Residual ethanol may interfere with downstream applications.
   13. Transfer the DNA mini column to a clean 1.5 mL microcentrifuge tube.
   14. Add 30-100 µL of Elution Buffer or sterile deionized water directly to the center of the column membrane.
       NOTE: The efficiency of eluting DNA from the DNA Mini column is dependent on pH. If using sterile deionized water, make sure that the pH is around 8.5.
   15. Let sit at room temperature for 1 minute.
   16. Centrifuge at maximum speed for 1 minute.
       NOTE: This represents approximately 70% of bound DNA. An optional second elution will yield any residual DNA, though at a lower concentration.
3. Verify the above plasmids using by double restriction enzyme digestion using EcoR I (15 U/µL) and Bam H1(15 U/µL)²².
   1. Perform the make-up steps on the ice. Prepare the reaction system of 1 µL of EcoR I (15 U), 1 µL of Bam H1 (15 U), 2 µL of 10x Buffer, 1 µg of plasmid; add ddH₂O to 20 µL of total volume, mix thoroughly. Incubate in a water bath at 37 °C for 2 hours.
   2. Separate the digested products by 1% agarose followed by electrophoresis and expose to UV light as before.
4. Dilute the concentrated plasmid into 2 x 10¹¹ copies/mL for the starting standard solution using ddH₂O. Take the plasmid (P2) containing a fragment of porcine DNA for example of copies calculating:
   1. Calculate the number of plasmids in 1 mL of starting standard solution: N = (2 x 10¹¹)/ (6.02 x 10²³) mol.
   2. Calculate the molecular weight of P2: M = 2810 bp x 650 D/bp = 2810 x 650 D (g/mol).
   3. Calculate the mass of P2: m=N*M= (2 x 10¹¹)/ (6.02 x 10²³) mol x 2810 x 650 g/mol.
   4. Calculate the volume of P2: V = m/C = [(2 x 10¹¹)/(6.02 x 10²³) x 2810 x 650] g/C. (C is the concentration of P2 (ng/µL), which is measured by a spectrophotometer).
5. Dilute the starting standard solution serially 10-fold into 2 x 10¹⁰ copies/mL, 2 x 10⁹ copies/mL, 2 x 10⁸ copies/mL, 2 x 10⁷ copies/mL…, and 2 x 10⁰ copies/mL for standard solution using ddH₂O. Vortex thoroughly during dilution.
6. Establish the standard curve.
   1. Prepare reaction system of qPCR (all steps are protected from light): Add the pre-mixed solution which contains 325 µL of qPCR Mix (Table of Materials), 13 µL of 5' primer, 13 µL of 3' primer, and 169 µL of ddH₂O into a 1.5 mL microcentrifuge tube, which was then thoroughly mixed and slightly centrifuged.
   2. Split the 40 µL above pre-mixed solution into an 8-tube strip and add 10 µL of standard DNA of different concentrations. Cap carefully, mix and centrifuge slightly.
   3. Put the 8-tube strip into the qPCR machine following the procedure shown in Figure 3.

5. Isolate circulating DNA from the blood samples

1. Using EDTA tubes, collect blood samples at different time points in the pig-to-monkey artery patch models and the pig-to-mouse cell transplantation models.
2. Collect blood samples of about 400 µL (from the pig-to-monkey artery patch models) or 100 µL (from the pig-to-mouse cell transplantation models) and transfer to 1.5 mL microcentrifuge tubes. Remove the blood cells from the blood samples by centrifugation at low temperature and high speed (3,000 x g, 4 °C, 5 min).
3. Transfer the supernatant to a new 1.5 mL microcentrifuge tube and remove the cell debris by centrifugation at 16,000 x g for 10 min.
4. Transfer the supernatant to a new 1.5 mL microcentrifuge tube. Extract the circulating DNA from the above supernatant using a commercial serum/circulating DNA extraction kit following protocol 2 of the isolating genomic DNA manufacturers’ protocol.
5. Condense the volume of circulating DNA to 40 µL and store at -20 °C.

6. Quantitation of circulating pig-specific DNA

1. Prepare the pre-mixed solution of 25 µL of qPCR Mix (Table of Materials), 1 µL of 5' primer, 1 µL of 3' primer, 10 µL of Sample DNA, and 13 µL of ddH₂O. Prepare the mix including 2 extra samples.
2. Split the 40 µL pre-mixed solution into an 8-tube strip, and add 10 µL of sample DNA, followed by careful capping. Prepare two more reactions than needed.
3. Put the 8-tube strip into the qPCR machine following the same procedure as the standard curve. It is better to run a standard first to make sure the reaction is critical for quantification in advance. The procedure of the reaction system of qPCR was shown in Figure 3.

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

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 Ⅰ	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