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-specific genes (Table 1) using the software of Primer 5.</li>
</ol>
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).
<ol>
	<li>Place 500 &#181;L of the whole blood of above samples into different microcentrifuge tubes, respectively.</li>
	<li>Add 20 &#181;L of protease K and 500 &#181;L of lysis buffer&#160;into the above microcentrifuge tubes, and then thoroughly shake and mix.</li>
	<li>Put these microcentrifuge tubes in a water bath at 56 &#176;C for 10 min and shake 2-3 times during this process until the solution becomes clear.</li>
	<li>Centrifuge briefly to remove the liquid beads from the inner wall of the tube covers. Add 500 &#181;L of anhydrous ethyl alcohol and shake thoroughly.</li>
	<li>Transfer the mixture into the adsorption column, centrifuge at 12,000 rpm (~13,400&#160;x g) for 2 min.</li>
	<li>Add 800 &#181;L of rinse solution to each adsorption column; centrifuge for 1 min at 12,000 rpm (&#160;~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.</li>
	<li>Transfer the adsorption columns to another clean centrifugal tube, add 50 &#181;L 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.</li>
	<li>Store the DNA solution at -20 &#176;C.</li>
</ol>
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.
<ol>
	<li>Prepare the pre-mixed solution of 12.5 &#181;L of 2x PCR premix&#160;(Table of Materials), 1 &#181;L of 5&#39; primer (10 &#181;M), 1 &#181;L of 3&#39; primer (10 &#181;M), and 8.5 &#181;L of ddH2O. Prepare the mix including 2 extra samples.</li>
	<li>Split 23 &#181;L of pre-mixed solution into 0.2&#160;mL microcentrifuge tubes, add 2 &#181;L of genomic DNA, and carefully cap the tubes. Then mix and centrifuge slightly.</li>
	<li>Place the 0.2&#160;mL microcentrifuge tubes into the PCR-cycler and perform the following: Denaturation: 95 &#176;C for 5 s; Annealing: 60 &#176;C for 30 s; extension:72 &#176;C for 30 s.</li>
	<li>Perform agarose electrophoresis as follows:
	<ol>
		<li>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 &#181;L of nucleic acid dye (Table of Materials) into the flask after it cools to about 70 &#176;C. Pour it into the plate along the edge slowly, and place it at room temperature until it solidifies into a gel.</li>
		<li>Add 5 &#181;L of sample and 2-Log DNA Ladder (0.1&#8211;10.0 kb) or Marker I (0.1&#8211;0.6 kb), which contain 1 &#181;L of 6x DNA loading buffer, into the agarose gel. Then electrophorese at 120 mA until the bands are separated.</li>
		<li>Visualize the agar gel containing DNA fragments with an ultraviolet imager.</li>
	</ol>
	</li>
	<li>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)</li>
</ol>
4. Standard curve of cpsDNA
<ol>
	<li>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 &#181;g/mL ampicillin. 
	Primer #4 in human/monkey cohort (forward: 5&#8242;-TTCAATCCCA CTTCTTCCACCTAA-3&#8242;, reverse: 5&#8242;-CTTCATTCCATCTTCATAATAAC CCTGT-3&#8242;) 
	Primer #11 for mouse model (forward: 5&#8242;-TGCCGTGGTTTCC GTTGCTTG-3&#8242;, reverse: 5&#8242;-TCACATTTGATGGTCGTCTTGTCGTC T-3&#8242;) 
	NOTE: Details of all the primers can be found in Table 1.</li>
	<li>Harvest the above plasmids following the protocol below.
	<ol>
		<li>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 &#176;C with vigorous shaking (300 rpm).</li>
		<li>Centrifuge at 10,000 x g for 1 minute at room temperature. Decant or aspirate and discard the culture media.</li>
		<li>Add 250 &#181;L 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.</li>
		<li>Transfer suspension into a new 1.5 mL microcentrifuge tube. Add 250 &#181;L 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.</li>
		<li>Add 350 &#181;L 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.</li>
		<li>Centrifuge at maximum speed (&#8805;13,000 x g) for 10 minutes. A compact white pellet will form. Promptly proceed to the next step.</li>
		<li>Insert a DNA mini column into a 2 mL collection tube.</li>
		<li>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.</li>
		<li>Centrifuge at maximum speed for 1 minute. Discard the filtrate and reuse the collection tube.</li>
		<li>Add 500 &#181;L 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.</li>
		<li>Add 700 &#181;L 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.
		<ol>
			<li>Optional: Repeat for a second DNA wash buffer wash step.</li>
		</ol>
		</li>
		<li>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.</li>
		<li>Transfer the DNA mini column to a clean 1.5 mL microcentrifuge tube.</li>
		<li>Add 30-100 &#181;L 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.</li>
		<li>Let sit at room temperature for 1 minute.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>Verify the above plasmids using by double restriction enzyme digestion using EcoR I (15 U/&#181;L) and Bam H1(15 U/&#181;L)22.
	<ol>
		<li>Perform the make-up steps on the ice. Prepare the reaction system of 1 &#181;L of EcoR I (15 U), 1 &#181;L of Bam H1 (15 U), 2 &#181;L of 10x Buffer, 1 &#181;g of plasmid; add ddH2O to 20 &#181;L of total volume, mix thoroughly. Incubate in a water bath at 37 &#176;C for 2 hours.</li>
		<li>Separate the digested products by 1% agarose followed by electrophoresis and expose to UV light as before.</li>
	</ol>
	</li>
	<li>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:
	<ol>
		<li>Calculate the number of plasmids in 1 mL of starting standard solution: N = (2 x 1011)/ (6.02 x 1023) mol.</li>
		<li>Calculate the molecular weight of P2: M = 2810 bp x 650 D/bp = 2810 x 650 D (g/mol).</li>
		<li>Calculate the mass of P2: m=N*M= (2 x 1011)/ (6.02 x 1023) mol x 2810 x 650 g/mol.</li>
		<li>Calculate the volume of P2: V = m/C = [(2 x 1011)/(6.02 x 1023) x 2810 x 650] g/C.&#160;(C is the concentration of P2 (ng/&#181;L), which is measured by a spectrophotometer).</li>
	</ol>
	</li>
	<li>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&#8230;, and 2 x 100 copies/mL for standard solution using ddH2O. Vortex thoroughly during dilution.</li>
	<li>Establish the standard curve.
	<ol>
		<li>Prepare reaction system of qPCR (all steps are protected from light): Add the pre-mixed solution which contains 325 &#181;L of qPCR Mix (Table of Materials), 13 &#181;L of 5&#39; primer, 13 &#181;L of 3&#39; primer, and 169 &#181;L of ddH2O into a 1.5 mL microcentrifuge tube, which was then thoroughly mixed and slightly centrifuged.</li>
		<li>Split the 40 &#181;L above pre-mixed solution into an 8-tube strip and add 10 &#181;L of standard DNA of different concentrations. Cap carefully, mix and centrifuge slightly.</li>
		<li>Put the 8-tube strip into the qPCR machine following the procedure shown in Figure 3.</li>
	</ol>
	</li>
</ol>
5. Isolate circulating DNA from the blood samples
<ol>
	<li>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.</li>
	<li>Collect blood samples of about 400 &#181;L (from the pig-to-monkey artery patch models) or 100 &#181;L (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 &#176;C, 5 min).</li>
	<li>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.</li>
	<li>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&#8217; protocol.</li>
	<li>Condense the volume of circulating DNA to 40 &#181;L and store at -20 &#176;C.</li>
</ol>
6. Quantitation of circulating pig-specific DNA
<ol>
	<li>Prepare the pre-mixed solution of 25 &#181;L of qPCR Mix (Table of Materials), 1 &#181;L of 5&#39; primer, 1 &#181;L of 3&#39; primer, 10 &#181;L of Sample DNA, and 13 &#181;L of ddH2O. Prepare the mix including 2 extra samples.</li>
	<li>Split the 40 &#181;L pre-mixed solution into an 8-tube strip, and add 10 &#181;L of sample DNA, followed by careful capping. Prepare two more reactions than needed.</li>
	<li>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.</li>
</ol>

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The authors report no conflicts of interest.

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><tbody><tr><td>&lt;a&gt;Agarose&lt;/a&gt;</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&amp;nbsp;Biosciences, Union City, CA, USA</td><td>MCT-150-C</td><td></td></tr><tr><td>0.2 mL PCR tube</td><td>Axygen&amp;nbsp;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,&amp;nbsp; China</td><td></td><td>8~10 weeks</td></tr><tr><td>Centrifuge</td><td>Thermo&amp;nbsp;Fisher&amp;nbsp;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&amp;ndash;10.0 kb</td></tr><tr><td>Marker I&amp;nbsp;</td><td>Tiangen, Beijing, China</td><td>MD101-02</td><td>0.1&amp;ndash;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>E.Z.N.A.Plasmid DNA Mini Kit I and E.Z.N.A. Plasmid DNA Mini Kit II</td><td>Omega Bio-tek, Norcross, GA, USA</td><td>D6942</td><td></td></tr><tr><td>D6943</td><td></td><td></td><td></td></tr><tr><td>EcoR I</td><td>Takara&amp;nbsp;Bio, Shiga, Japan</td><td>&amp;nbsp;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 ?</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&amp;nbsp;Bio, Shiga, Japan</td><td>RR901A</td><td></td></tr><tr><td>pMD19-T plasmid</td><td>Takara&amp;nbsp;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...

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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 ...

quantification-circulating-pig-specific-dna-blood-xenotransplantation

Research

JoVE Journal

Biology

3.9K Views.  Shenzhen University School of Medicine, First Affiliated Hospital of Shenzhen University, Shenzhen Second People's Hospital. Xenotransplantatie is een haalbare methode om te behandelen Echter, effectieve monitoring van de immuunafstoting van xenotransplantatie is een probleem voor artsen en de onderzoekers. Dit protocol is een eenvoudige, handige, lage kosten en minder invasieve methode om de immuunafstoting van de xenotransmutatie te controleren. Deze techniek kan worden gebruikt in xenotransplantatie veld, met inbegrip van pig-to-human eilandje transplantatie, niertransplantatie, levertransplantatie en harttransp...

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

Labeling of Breast Cancer Patient-derived Xenografts with Traceable Reporters for Tumor Growth and Metastasis Studies

The use of preclinical models to study tumor biology and response to treatment is central to cancer research. Long-established human cell lines, and many transgenic mouse models, often fail to recapitulate the key aspects of human malignancies. Thus, alternative models that better represent the heterogeneity of patients' tumors and their metastases are being developed. Patient-derived xenograft (PDX) models in which surgically resected tumor samples are engrafted into immunocompromised mice have become an attractive alternative as they can be transplanted through multiple generations,and more efficiently reflect tumor heterogeneity than xenografts derived from human cancer cell lines. A limitation to the use of PDXs is that they are difficult to transfect or transduce to introduce traceable reporters or to manipulate gene expression. The current protocol describes methods to transduce dissociated tumor cells from PDXs with high transduction efficiency, and the use of labeled PDXs for experimental models of breast cancer metastases. The protocol also demonstrates the use of labeled PDXs in experimental metastasis models to study the organ-colonization process of the metastatic cascade. Metastases to different organs can be easily visualized and quantified using bioluminescent imaging in live animals, or GFP expression during dissection and in excised organs. These methods provide a powerful tool to extend the use of multiple types of PDXs to metastasis research.

We describe a method for stable labeling of patient-derived xenografts (PDXs) with lentiviral particles expressing green-fluorescent protein and luciferase reporters. This method allows for tracking the growth of PDXs at the primary site, as well as detecting spontaneous and experimental metastases using in vivo imaging systems.

The use of preclinical models to study tumor biology and response to treatment is central to cancer research. Long-established human cell lines, and many ...

Cancer Research

Patient-derived Heterogeneous Xenograft Model of Pancreatic Cancer Using Zebrafish Larvae as Hosts for Comparative Drug Assessment

Patient-derived tumor xenograft (PDX) and cell-derived tumor xenograft (CDX) are important techniques for preclinical assessment, medication guidance and basic cancer researches. Generations of PDX models in traditional host mice are time-consuming and only working for a small proportion of samples. Recently, zebrafish PDX (zPDX) has emerged as a unique host system, with the characteristics of small-scale and high efficiency. Here, we describe an optimized methodology for generating a dual fluorescence-labeled tumor xenograft model for comparative chemotherapy assessment in zPDX models. Tumor cells and fibroblasts were enriched from freshly-harvested or frozen pancreatic cancer tissue at different culture conditions. Both cell groups were labeled by lentivirus expressing green or red fluorescent proteins, as well as an anti-apoptosis gene BCL2L1. The transfected cells were pre-mixed and co-injected into the 2 dpf larval zebrafish that were then bred in modified E3 medium at 32 &#176;C. The xenograft models were treated by chemotherapy drugs and/or BCL2L1 inhibitor, and the viabilities of both tumor cells and fibroblasts were investigated simultaneously. In summary, this protocol allows researchers to quickly generate a large amount of zPDX models with a heterogeneous tumor microenvironment and provides a longer observation window and a more precise quantitation in assessing the efficiency of drug candidates.

This protocol describes optimization procedures in a virus-based dual fluorescence-labeled tumor xenograft model using larval zebrafish as hosts. This heterogeneous xenograft model mimics the tissue composition of pancreatic cancer microenvironment in vivo and serves as a more precise tool for assessing drug responses in personalized zPDX (zebrafish patient-derived xenograft) models.

Patient-derived tumor xenograft (PDX) and cell-derived tumor xenograft (CDX) are important techniques for preclinical assessment, medication guidance and ...

piggyBac Transposon System Modification of Primary Human T Cells

The piggyBac transposon system is naturally active, originally derived from the cabbage looper moth1,2. This non-viral system is plasmid based, most commonly utilizing two plasmids with one expressing the piggyBac transposase enzyme and a transposon plasmid harboring the gene(s) of interest between inverted repeat elements which are required for gene transfer activity. PiggyBac mediates gene transfer through a "cut and paste" mechanism whereby the transposase integrates the transposon segment into the genome of the target cell(s) of interest. PiggyBac has demonstrated efficient gene delivery activity in a wide variety of insect1,2, mammalian3-5, and human cells6 including primary human T cells7,8. Recently, a hyperactive piggyBac transposase was generated improving gene transfer efficiency9,10.
Human T lymphocytes are of clinical interest for adoptive immunotherapy of cancer11. Of note, the first clinical trial involving transposon modification of human T cells using the Sleeping beauty transposon system has been approved12. We have previously evaluated the utility of piggyBac as a non-viral methodology for genetic modification of human T cells. We found piggyBac to be efficient in genetic modification of human T cells with a reporter gene and a non-immunogenic inducible suicide gene7. Analysis of genomic integration sites revealed a lack of preference for integration into or near known proto-oncogenes13. We used piggyBac to gene-modify cytotoxic T lymphocytes to carry a chimeric antigen receptor directed against the tumor antigen HER2, and found that gene-modified T cells mediated targeted killing of HER2-positive tumor cells in vitro and in vivo in an orthotopic mouse model14. We have also used piggyBac to generate human T cells resistant to rapamycin, which should be useful in cancer therapies where rapamycin is utilized15.
Herein, we describe a method for using piggyBac to genetically modify primary human T cells. This includes isolation of peripheral blood mononuclear cells (PBMCs) from human blood followed by culture, gene modification, and activation of T cells. For the purpose of this report, T cells were modified with a reporter gene (eGFP) for analysis and quantification of gene expression by flow cytometry.
PiggyBac can be used to modify human T cells with a variety of genes of interest. Although we have used piggyBac to direct T cells to tumor antigens14, we have also used piggyBac to add an inducible safety switch in order to eliminate gene modified cells if needed7. The large cargo capacity of piggyBac has also enabled gene transfer of a large rapamycin resistant mTOR molecule (15 kb)15. Therefore, we present a non-viral methodology for stable gene-modification of primary human T cells for a wide variety of purposes.

piggyBac Transposon System Modification of Primary Human T Cells

We describe a method to genetically modify primary human T cells with a transgene using the non-viral piggyBac transposon system. T cells modified to using the piggyBac transposon system exhibit stable transgene expression.

The piggyBac transposon system is naturally active, originally derived from the cabbage looper moth1,2. This non-viral system is plasmid based, most ...

Immunology and Infection

Surgical Angiogenesis in Porcine Tibial Allotransplantation: A New Large Animal Bone Vascularized Composite Allotransplantation Model

Segmental bone loss resulting from trauma, infection malignancy and congenital anomaly remains a major reconstructive challenge. Current therapeutic options have significant risk of failure and substantial morbidity.
Use of bone vascularized composite allotransplantation (VCA) would offer both a close match of resected bone size and shape and the healing and remodeling potential of living bone. At present, life-long drug immunosuppression (IS) is required. Organ toxicity, opportunistic infection and neoplasm risks are of concern to treat such non-lethal indications.
We have previously demonstrated that bone and joint VCA viability may be maintained in rats and rabbits without the need of long-term-immunosuppression by implantation of recipient derived vessels within the VCA. It generates an autogenous, neoangiogenic circulation with measurable flow and active bone remodeling, requiring only 2 weeks of IS. As small animals differ from man substantially in anatomy, bone physiology and immunology, we have developed a porcine bone VCA model to evaluate this technique before clinical application is undertaken. Miniature swine are currently widely used for allotransplantation research, given their immunologic, anatomic, physiologic and size similarities to man. Here, we describe a new porcine orthotopic tibial bone VCA model to test the role of autogenous surgical angiogenesis to maintain VCA viability.
The model reconstructs segmental tibial bone defects using size- and shape-matched allogeneic tibial bone segments, transplanted across a major swine leukocyte antigen (SLA) mismatch in Yucatan miniature swine. Nutrient vessel repair and implantation of recipient derived autogenous vessels into the medullary canal of allogeneic tibial bone segments is performed in combination with simultaneous short-term IS. This permits a neoangiogenic autogenous circulation to develop from the implanted tissue, maintaining flow through the allogeneic nutrient vessels for a short time. Once established, the new autogenous circulation maintains bone viability following cessation of drug therapy and subsequent nutrient vessel thrombosis.

Currently any kind of vascularized composite allotransplantation depends on long-term-immunosuppression, difficult to support for non-life-critical indications. We present a new porcine tibial VCA model that can be used to study bone VCA and demonstrate the use of surgical angiogenesis to maintain bone viability without the need of long-term immune-modulation.

Segmental bone loss resulting from trauma, infection malignancy and congenital anomaly remains a major reconstructive challenge. Current therapeutic ...

Medicine

Optimized Interferon-gamma ELISpot Assay to Measure T Cell Responses in the Guinea Pig Model after Vaccination

The guinea pig has played a pivotal role as a relevant small animal model in the development of vaccines for infectious diseases such as tuberculosis, influenza, diphtheria, and viral hemorrhagic fevers. We have demonstrated that plasmid-DNA (pDNA) vaccine delivery into the skin elicits robust humoral responses in the guinea pig. However, the use of this animal to model immune responses was somewhat limited in the past due to the lack of available reagents and protocols to study T cell responses. T cells play a pivotal role in both immunoprophylactic and immunotherapeutic mechanisms. Understanding T cell responses is crucial for the development of infectious disease and oncology vaccines and accommodating delivery devices. Here we describe an interferon-gamma (IFN-&#947;) enzyme-linked immunospot (ELISpot) assay for guinea pig peripheral blood mononuclear cells (PBMCs). The assay enables researchers to characterize vaccine-specific T-cell responses in this important rodent model. The ability to assay cells isolated from the peripheral blood provides the opportunity to track immunogenicity in individual animals.

The development of the interferon-gamma ELISpot assay for guinea pig PBMCs allows the characterization of T-cell responses in this highly relevant model for studying infectious diseases. We have applied the assay to measure T cell responses associated with intradermal delivery of DNA vaccines.

The guinea pig has played a pivotal role as a relevant small animal model in the development of vaccines for infectious diseases such as tuberculosis, ...

Expression of Exogenous Cytokine in Patient-derived Xenografts via Injection with a Cytokine-transduced Stromal Cell Line

Patient-derived xenograft (PDX) mice are produced by transplanting human cells into immune deficient mice. These models are an important tool for studying the mechanisms of normal and malignant hematopoiesis and are the gold standard for identifying effective chemotherapies for many malignancies. PDX models are possible because many of the mouse cytokines also act on human cells. However, this is not the case for all cytokines, including many that are critical for studying normal and malignant hematopoiesis in human cells. Techniques that engineer mice to produce human cytokines (transgenic and knock-in models) require significant expense before the usefulness of the model has been demonstrated. Other techniques are labor intensive (injection of recombinant cytokine or lentivirus) and in some cases require high levels of technical expertise (hydrodynamic injection of DNA). This report describes a simple method for generating PDX mice that have exogenous human cytokine (TSLP, thymic stromal lymphopoietin) via weekly intraperitoneal injection of stroma that have been transduced to overexpress this cytokine. Use of this method provides an in vivo source of continuous cytokine production that achieves physiological levels of circulating human cytokine in the mouse. Plasma levels of human cytokine can be varied based on the number of stromal cells injected, and cytokine production can be initiated at any point in the experiment. This method also includes cytokine-negative control mice that are similarly produced, but through intraperitoneal injection of stroma transduced with a control vector. We have previously demonstrated that leukemia cells harvested from TSLP-expressing PDX, as compared to control PDX, exhibit a gene expression pattern more like the original patient sample. Together the cytokine-producing and cytokine-negative PDX mice produced by this method provide a model system that we have used successfully to study the role of TSLP in normal and malignant hematopoiesis.

Described here is a method for producing exogenous cytokine in patient-derived xenograft (PDX) mice via weekly intraperitoneal injection of a cytokine-transduced stromal cell line. This method broadens the utility of PDX and provides the option for transient or sustained exogenous cytokine delivery in a multitude of PDX models.

Patient-derived xenograft (PDX) mice are produced by transplanting human cells into immune deficient mice. These models are an important tool for studying ...

Induction and Scoring of Graft-Versus-Host Disease in a Xenogeneic Murine Model and Quantification of Human T Cells in Mouse Tissues using Digital PCR

Acute graft-versus-host disease (GVHD) is a significant limitation for patients receiving hematopoietic stem cell transplant as therapy for hematological deficiencies and malignancies. Acute GVHD occurs when donor T cells recognize host tissues as a foreign antigen and mount an immune response to the host. Current treatments involve toxic immunosuppressive drugs that render patients susceptible to infection and recurrence. Thus, there is ongoing research to provide an acute GVHD therapy that can effectively target donor T cells and reduce side effects. Much of this pre-clinical work uses the xenogenic GVHD (xenoGVHD) murine model that allows for testing of immunosuppressive therapies on human cells rather than murine cells in an in vivo system. This protocol outlines how to induce xenoGVHD and how to blind and standardize clinical scoring to ensure consistent results. Additionally, this protocol describes how to use digital PCR to detect human T cells in mouse tissues, which can subsequently be used to quantify efficacy of tested therapies. The xenoGVHD model not only provides a model to test GVHD therapies but any therapy that can suppress human T cells, which could then be applied to many inflammatory diseases.

Here, we present a protocol to induce and score disease in a xenogeneic graft-versus-host disease (xenoGVHD) model. xenoGVHD provides an in vivo model to study immunosuppression of human T cells. Additionally, we describe how to detect human T cells in tissues with digital PCR as a tool to quantify immunosuppression.

Acute graft-versus-host disease (GVHD) is a significant limitation for patients receiving hematopoietic stem cell transplant as therapy for hematological ...

Lentiviral Vector-mediated Gene Therapy of Hepatocytes Ex Vivo for Autologous Transplantation in Swine

Gene therapy is an ideal choice to cure many inborn errors of metabolism of the liver. Ex-vivo, lentiviral vectors have been used successfully in the treatment of many hematopoietic diseases in humans, as their use offers stable transgene expression due to the vector&#39;s ability to integrate into the host genome. This method demonstrates the application of ex vivo gene therapy of hepatocytes to a large animal model of hereditary tyrosinemia type I. This process consists of 1) isolation of primary hepatocytes from the autologous donor/recipient animal, 2) ex vivo gene delivery via hepatocyte transduction with a lentiviral vector, and 3) autologous transplant of corrected hepatocytes via portal vein injection. Success of the method generally relies upon efficient and sterile removal of the liver resection, careful handling of the excised specimen for isolation of viable hepatocytes sufficient for re-engrafting, high-percentage transduction of the isolated cells, and aseptic surgical procedures throughout to prevent infection. Technical failure at any of these steps will result in low yield of viable transduced hepatocytes for autologous transplant or infection of the donor/recipient animal. The pig model of human type 1 hereditary tyrosinemia (HT-1) chosen for this approach is uniquely amenable to such a method, as even a small percentage of engraftment of corrected cells will lead to repopulation of the liver with healthy cells based on a powerful selective advantage over native-diseased hepatocytes. Although this growth selection will not be true for all indications, this approach is a foundation for expansion into other indications and allows for manipulation of this environment to address additional diseases, both within the liver and beyond, while controlling for exposure to viral vector and opportunity for off-target toxicity and tumorigenicity.

Lentiviral Vector-mediated Gene Therapy of Hepatocytes Ex Vivo for Autologous Transplantation in Swine

This protocol is intended to describe porcine hepatocyte isolation and ex vivo gene delivery to cure models of metabolic diseases via autologous cell transplantation. Although this particular model enjoys unique advantages that favor successful therapy, the application is a relevant foundation to address additional diseases and indications.

Gene therapy is an ideal choice to cure many inborn errors of metabolism of the liver. Ex-vivo, lentiviral vectors have been used successfully in the ...

A Porcine Heterotopic Heart Transplantation Protocol for Delivery of Therapeutics to a Cardiac Allograft

Cardiac transplantation is the gold standard treatment for end-stage heart failure. However, it remains limited by the number of available donor hearts and complications such as primary graft dysfunction and graft rejection. The recent clinical use of an ex vivo perfusion device in cardiac transplantation introduces a unique opportunity for treating cardiac allografts with therapeutic interventions to improve function and avoid deleterious recipient responses. Establishing a translational, large-animal model for therapeutic delivery to the entire allograft is essential for testing novel therapeutic approaches in cardiac transplantation. The porcine, heterotopic heart transplantation model in the intraabdominal position serves as an excellent model for assessing the effects of novel interventions and the immunopathology of graft rejection. This model additionally offers long-term survival for the pig, given that the graft is not required to maintain the recipient's circulation. The aim of this protocol is to provide a reproducible and robust approach for achieving ex vivo delivery of a therapeutic to the entire cardiac allograft prior to transplantation and provide technical details to perform a survival heterotopic transplant of the ex vivo perfused heart.

We present a protocol for utilizing a normothermic ex vivo sanguinous perfusion system for the delivery of therapeutics to an entire cardiac allograft in a porcine heterotopic heart transplant model.

Cardiac transplantation is the gold standard treatment for end-stage heart failure. However, it remains limited by the number of available donor hearts ...

Porcine Liver Transplantation Without Veno-Venous Bypass As an Extended Criteria Donor Model

Liver transplantation is regarded as the gold standard for the treatment of a variety of fatal hepatic diseases. However, unsolved issues of chronic graft failure, ongoing organ donor shortages, and the increased use of marginal grafts call for the improvement of current concepts, such as the implementation of organ machine perfusion. In order to evaluate new methods of graft reconditioning and modulation, translational models are required. With respect to anatomical and physiological similarities to humans and recent progress in the field of xenotransplantation, pigs have become the main large animal species used in transplantation models. After the initial introduction of a porcine orthotopic liver transplant model by Garnier et al. in 1965, several modifications have been published over the past 60 years.
Due to specifies-specific anatomical traits, a veno-venous bypass during the anhepatic phase is regarded as a necessity to reduce intestinal congestion and ischemia resulting in hemodynamic instability and perioperative mortality. However, the implementation of a bypass increases the technical and logistical complexity of the procedure. Furthermore, associated complications such as air embolism, hemorrhage, and the need for a simultaneous splenectomy have been reported previously.
In this protocol, we describe a model of porcine orthotopic liver transplantation without the use of a veno-venous bypass. The engraftment of donor livers after static cold storage of 20 h - simulating extended criteria donor conditions - demonstrates that this simplified approach can be performed without significant hemodynamic alterations or intraoperative mortality and with regular uptake of liver function (as defined by bile production and liver-specific CYP1A2 metabolism). The success of this approach is ensured by an optimized surgical technique and a sophisticated anesthesiologic volume and vasopressor management.
This model should be of special interest for workgroups focusing on the immediate postoperative course, ischemia-reperfusion injury, associated immunological mechanisms, and the reconditioning of extended criteria donor organs.

In this protocol, a model of porcine orthotopic liver transplantation after static cold storage of donor organs for 20 h without the use of a veno-venous bypass during engraftment is described. The approach uses a simplified surgical technique with minimization of the anhepatic phase and sophisticated volume and vasopressor management.

Liver transplantation is regarded as the gold standard for the treatment of a variety of fatal hepatic diseases. However, unsolved issues of chronic graft ...

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

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