Name: quantification of circulating pig-specific dna in the blood of a xenotransplantation model
Uploaded: 2020-09-22T14:00:00
Description: Watch this Scientific Journal Video about Quantification of Circulating Pig-Specific DNA in the Blood of a Xenotransplantation Model at JoVE.com

This work were supported by grants from National Key R&#38;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).

All experiments were performed in accordance with the relevant guidelines and regulations of the Institutional Review Board of Shenzhen Second People&#39;s Hospital, First Af&#64257;liated Hospital of Shenzhen University.
1. Design porcine specific primers
<ol>
	<li>Perform whole-genome BLAST analysis to identify porcine specific genes that were different from those of humans, monkeys or mouse, using the software NCBI&#160;(www.ncbi.nlm.nih.gov).</li>
	<li>Design primers according to 19 pig-...

<ol>
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A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-invasive+approaches+for+the+diagnosis+of+acute+cardiac+allograft+rejection.">Non-invasive approaches for the diagnosis of acute cardiac allograft rejection.</a> Heart. 99 (7), 445-453 (2013).</li><li>Mandel, P., Metais, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Les+acides+nucl&#233;iques+du+plasma+sanguin+chez+l'homme.">Les acides nucl&#233;iques du plasma sanguin chez l'homme.</a> Comptes Rendus des Seances de l Academie des Sciences. 142 (3-4), 241-243 (1948).</li><li>Okkonen, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasma+cell-free+DNA+in+patients+needing+mechanical+ventilation.">Plasma cell-free DNA in patients needing mechanical ventilation.</a> Critical Care. 15 (4), 196 (2011).</li><li>Wagner, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Free+DNA--new+potential+analyte+in+clinical+laboratory+diagnostics.">Free DNA--new potential analyte in clinical laboratory diagnostics.</a> Biochemia Medica. 22 (1), 24-38 (2012).</li><li>Schwarzenbach, H., Nishida, N., Calin, G. 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R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Universal+noninvasive+detection+of+solid+organ+transplant+rejection.">Universal noninvasive detection of solid organ transplant rejection.</a> Proceedings of the National Academy of Sciences of the United States of America. 108 (15), 6229-6234 (2011).</li><li>De Vlaminck, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Noninvasive+monitoring+of+infection+and+rejection+after+lung+transplantation.">Noninvasive monitoring of infection and rejection after lung transplantation.</a> Proceedings of the National Academy of Sciences of the United States of America. 112 (43), 13336-13341 (2015).</li><li>Zhou, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circulating+pig-specific+DNA+as+a+novel+biomarker+for+monitoring+xenograft+rejection.">Circulating pig-specific DNA as a novel biomarker for monitoring xenograft rejection.</a> Xenotransplantation. 26 (4), 12522 (2019).</li><li>Huo, Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circulating+mi+RNA+or+circulating+DNA+&#8212;Potential+biomarkers+for+organ+transplant+rejection.">Circulating mi RNA or circulating DNA &#8212;Potential biomarkers for organ transplant rejection.</a> Xenotransplantation. 26 (1), 12444 (2019).</li><li>Gadi, V. 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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...

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. Currently, clinical monitoring of immune rejection depends mainly on the patient&#39;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 increased13,14 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 cancers17,18,19. High through-put sequencing of donor-derived circulating DNA is reliable for the detection of rejection after organ transplantation20,21. 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&#39;s blood post-xenotransplantation because of xeno-rejection. CpsDNA could be precisely quantified by qPCR with species-specific primers in the recipient&#8217;s blood. Previously, we have demonstrated the rationale and feasibility of cpsDNA as a biomarker for xenotransplantation22,23. 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&#160;qPCR.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr >
			<td ><a>Agarose</a></td>
			<td >Biowest, Barcelona, Spain</td>
			<td >111860</td>
			<td ></td>
		</tr>
		<tr >
			<td >BamHI-HF</td>
			<td >New England Biolabs, Ipswich, Mass, USA</td>
			<td >R3136S</td>
			<td ></td>
		</tr>
		<tr >
			<td >1.5 mL microcentrifuge tube</td>
			<td >Axygen&#160;Biosciences, Union City, CA, USA</td>
			<td >MCT-150-C</td>
			<td ></td>
		</tr>
		<tr >
			<td >0.2 mL PCR tube</td>
			<td >Axygen&#160;Biosciences, Union City, CA, USA</td>
			<td >PCR-02-C</td>
			<td ></td>
		</tr>
		<tr >
			<td >C57BL/6 Mice</td>
			<td >Medical Animal Center of Guangdong Province,&#160; China</td>
			<td ></td>
			<td >8~10 weeks</td>
		</tr>
		<tr >
			<td >Centrifuge</td>
			<td >Thermo&#160;Fisher&#160;Scientific, Walt- ham, MA, USA</td>
			<td >Micro 21R</td>
			<td ></td>
		</tr>
		<tr >
			<td >2-Log DNA Ladder</td>
			<td >New England Biolabs, Ipswich, Mass, USA</td>
			<td >N3200S</td>
			<td >0.1&#8211;10.0 kb</td>
		</tr>
		<tr >
			<td >Marker I&#160;</td>
			<td >Tiangen, Beijing, China</td>
			<td >MD101-02</td>
			<td >0.1&#8211;0.6 kb</td>
		</tr>
		<tr >
			<td >DNA Mini Column(HiBind DNA Mini Columns)</td>
			<td >Omega Bio-tek, Norcross, GA, USA</td>
			<td >DNACOL-01</td>
			<td ></td>
		</tr>
		<tr >
			<td >DNA loading buffer</td>
			<td >Solarbio, Beijing, China</td>
			<td >D1010</td>
			<td ></td>
		</tr>
		<tr >
			<td rowspan="2" >E.Z.N.A.Plasmid DNA Mini Kit I and E.Z.N.A. Plasmid DNA Mini Kit II</td>
			<td rowspan="2" >Omega Bio-tek, Norcross, GA, USA</td>
			<td >D6942</td>
			<td rowspan="2" ></td>
		</tr>
		<tr >
			<td >D6943</td>
		</tr>
		<tr >
			<td >EcoR I</td>
			<td >Takara&#160;Bio, Shiga, Japan</td>
			<td >&#160;1040S</td>
			<td ></td>
		</tr>
		<tr >
			<td >Female Bama mini pigs</td>
			<td >BGI Ark Biotechnology, Shenzhen, China</td>
			<td ></td>
			<td >2~4 months</td>
		</tr>
		<tr >
			<td >Genomic DNA Extraction Kit &#8544;</td>
			<td >Tiangen, Beijing, China</td>
			<td >DP304-02</td>
			<td ></td>
		</tr>
		<tr >
			<td >SYBR Green Realtime PCR Master Mix</td>
			<td >Toyobo, Osaka, Japan</td>
			<td >QPK-201</td>
			<td ></td>
		</tr>
		<tr >
			<td >Gel Doc XR</td>
			<td >Bio-Rad, Hercules, USA</td>
			<td ></td>
			<td ></td>
		</tr>
		<tr >
			<td >Male cynomolgus monkeys</td>
			<td >Guangdong Landau Biotechnology, Guangzhou, China</td>
			<td ></td>
			<td >8 years</td>
		</tr>
		<tr >
			<td >Nucleic acid dye(Gelred)</td>
			<td >Biotium, Fremont, USA</td>
			<td >42003</td>
			<td ></td>
		</tr>
		<tr >
			<td >polymerase(Premix Taq)</td>
			<td >Takara&#160;Bio, Shiga, Japan</td>
			<td >RR901A</td>
			<td ></td>
		</tr>
		<tr >
			<td >pMD19-T plasmid</td>
			<td >Takara&#160;Bio, Shiga, Japan</td>
			<td >D102A</td>
			<td ></td>
		</tr>
		<tr >
			<td >qPCR machine</td>
			<td >Applied Biosystems QSDx, Waltham, USA</td>
			<td ></td>
			<td ></td>
		</tr>
		<tr >
			<td >Serum/Circulating DNA Extraction Kit</td>
			<td >Tiangen, Beijing, China</td>
			<td >DP339</td>
			<td ></td>
		</tr>
		<tr >
			<td >TAE</td>
			<td >sangon Biotech, Shanghai, China</td>
			<td >B548101</td>
			<td ></td>
		</tr>
	</tbody>
</table>

quantification of circulating pig-specific dna in the blood of a xenotransplantation model

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.

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...

Watch this Scientific Journal Video about Quantification of Circulating Pig-Specific DNA in the Blood of a Xenotransplantation Model at JoVE.com

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

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.

Xenotransplantation is a feasible method to treat organ failure. However, how to effectively monitor the immune rejection of xenotransplantation is a ...

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