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 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 ...

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. 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.

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

Automated Microfluidic Blood Lysis Protocol for Enrichment of Circulating Nucleated Cells

In this report the protocol for an automated microfluidic blood lysis device is detailed. Circulating nucleated cells (CNCs), including leukocytes and endothelial cells, provide an ideal platform for an updated status on the immune condition of an individual. The microfluidic protocol allows for enrichment of CNCs without selective cell loss and sample preparation variability due to user-mediated steps. Briefly, the protocol includes device fabrication, sample collection, device setup, and running blood through the microfluidic chamber. Within the device whole blood is rapidly mixed with deionized water for approximately 10 seconds in a 50 micron x 150 micron microfluidic channel. In this time span erythrocytes are lysed due to hypotonic conditions. Herringbone structures on the bottom of the channel ensure thorough mixing and exposure of cells to a constant environment. Remaining cells are returned to isotonic conditions at the exit of the device, fixed using 2% paraformaldehyde, centrifuged to separate erythrocyte debris from CNCs, and suspended in flow buffer for staining and analysis by flow cytometry. Results show clean flow cytometry scatter plots with CNC populations saved. Significance of this device and protocol comes in the study and understanding of disease pathogenesis by analysis of CNC populations. Hence, automation, effectiveness, and simplicity of the microfluidic protocol are demonstrated.

An automated microfluidic device was developed for circulating nucleated cell enrichment from peripheral blood via erythrocyte lysis that ensures isolation of high quality sample without cell loss.

In this report the protocol for an automated microfluidic blood lysis device is detailed. Circulating nucleated cells (CNCs), including leukocytes and ...

Quantification of &gamma;H2AX Foci in Response to Ionising Radiation

DNA double-strand breaks (DSBs), which are induced by either endogenous metabolic processes or by exogenous sources, are one of the most critical DNA lesions with respect to survival and preservation of genomic integrity. An early response to the induction of DSBs is phosphorylation of the H2A histone variant, H2AX, at the serine-139 residue, in the highly conserved C-terminal SQEY motif, forming &gamma;H2AX1. Following induction of DSBs, H2AX is rapidly phosphorylated by the phosphatidyl-inosito 3-kinase (PIKK) family of proteins, ataxia telangiectasia mutated (ATM), DNA-protein kinase catalytic subunit and ATM and RAD3-related (ATR)2. Typically, only a few base-pairs (bp) are implicated in a DSB, however, there is significant signal amplification, given the importance of chromatin modifications in DNA damage signalling and repair. Phosphorylation of H2AX mediated predominantly by ATM spreads to adjacent areas of chromatin, affecting approximately 0.03% of total cellular H2AX per DSB2,3. This corresponds to phosphorylation of approximately 2000 H2AX molecules spanning ~2 Mbp regions of chromatin surrounding the site of the DSB and results in the formation of discrete &gamma;H2AX foci which can be easily visualized and quantitated by immunofluorescence microscopy2. The loss of &gamma;H2AX at DSB reflects repair, however, there is some controversy as to what defines complete repair of DSBs; it has been proposed that rejoining of both strands of DNA is adequate however, it has also been suggested that re-instatement of the original chromatin state of compaction is necessary4-8. The disappearence of &gamma;H2AX involves at least in part, dephosphorylation by phosphatases, phosphatase 2A and phosphatase 4C5,6. Further, removal of &gamma;H2AX by redistribution involving histone exchange with H2A.Z has been implicated7,8. Importantly, the quantitative analysis of &gamma;H2AX foci has led to a wide range of applications in medical and nuclear research. Here, we demonstrate the most commonly used immunofluorescence method for evaluation of initial DNA damage by detection and quantitation of &gamma;H2AX foci in &gamma;-irradiated adherent human keratinocytes9.

Quantification of &amp;#947;H2AX Foci in Response to Ionising Radiation

Quantification of DNA double-strand streaks using &gamma;H2AX formation as a molecular marker has become an invaluable tool in radiation biology. Here we demonstrate the use of an immunofluorescence assay for quantification of &gamma;H2AX foci after exposure of cells to radiation.

DNA double-strand breaks (DSBs), which are induced by either endogenous metabolic processes or by exogenous sources, are one of the most critical DNA ...

Xenotransplantation of Human Stem Cells into the Chicken Embryo

The chicken embryo is a classical animal model for studying normal embryonic and fetal development and for xenotransplantation experiments to study the behavior of cells in a standardized in vivo environment. The main advantages of the chicken embryo include low cost, high accessibility, ease of surgical manipulation and lack of a fully developed immune system. Xenotransplantation into chicken embryos can provide valuable information about cell proliferation, differentiation and behavior, the responses of cells to signals in defined embryonic tissue niches, and tumorigenic potential. Transplanting cells into chicken embryos can also be a step towards transplantation experiments in other animal models. Recently the chicken embryo has been used to evaluate the neurogenic potential of human stem and progenitor cells following implantation into neural anlage1-6. In this video we document the entire procedure for transplanting human stem cells into the developing central nervous system of the chicken embryo. The procedure starts with incubation of fertilized eggs until embryos of the desired age have developed. The eggshell is then opened, and the embryo contrasted by injecting dye between the embryo and the yolk. Small lesions are made in the neural tube using microsurgery, creating a regenerative site for cell deposition that promotes subsequent integration into the host tissue. We demonstrate injections of human stem cells into such lesions made in the part of the neural tube that forms the hindbrain and the spinal cord, and into the lumen of the part of the neural tube that forms the brain. Systemic injections into extraembryonic veins and arteries are also demonstrated as an alternative way to deliver cells to vascularized tissues including the central nervous system. Finally we show how to remove the embryo from the egg after several days of further development and how to dissect the spinal cord free for subsequent physiological, histological or biochemical analyses.

In this paper we present a method for transplanting human stem cells into various regions of the central nervous system of the chicken embryo. This provides an in vivo model for assessing the proliferation and differentiation of various types of human stem cells in embryonic tissue environments.

The chicken embryo is a classical animal model for studying normal embryonic and fetal development and for xenotransplantation experiments to study the ...

Whole-mount Immunohistochemical Analysis for Embryonic Limb Skin Vasculature: a Model System to Study Vascular Branching Morphogenesis in Embryo

Whole-mount immunohistochemical analysis for imaging the entire vasculature is pivotal for understanding the cellular mechanisms of branching morphogenesis. We have developed the limb skin vasculature model to study vascular development in which a pre-existing primitive capillary plexus is reorganized into a hierarchically branched vascular network. Whole-mount confocal microscopy with multiple labelling allows for robust imaging of intact blood vessels as well as their cellular components including endothelial cells, pericytes and smooth muscle cells, using specific fluorescent markers. Advances in this limb skin vasculature model with genetic studies have improved understanding molecular mechanisms of vascular development and patterning. The limb skin vasculature model has been used to study how peripheral nerves provide a spatial template for the differentiation and patterning of arteries. This video article describes a simple and robust protocol to stain intact blood vessels with vascular specific antibodies and fluorescent secondary antibodies, which is applicable for vascularized embryonic organs where we are able to follow the process of vascular development.

We introduce a whole-mount immunohistochemistry and laser scanning confocal microscopy with multiple labelling for analyzing intricate vascular network formation in mouse embryonic limb skin.

Whole-mount immunohistochemical analysis for imaging the entire vasculature is pivotal for understanding the cellular mechanisms of branching ...

Visualization of DNA Replication in the Vertebrate Model System DT40 using the DNA Fiber Technique

Maintenance of replication fork stability is of utmost importance for dividing cells to preserve viability and prevent disease. The processes involved not only ensure faithful genome duplication in the face of endogenous and exogenous DNA damage but also prevent genomic instability, a recognized causative factor in tumor development.
Here, we describe a simple and cost-effective fluorescence microscopy-based method to visualize DNA replication in the avian B-cell line DT40. This cell line provides a powerful tool to investigate protein function in vivo by reverse genetics in vertebrate cells1. DNA fiber fluorography in DT40 cells lacking a specific gene allows one to elucidate the function of this gene product in DNA replication and genome stability. Traditional methods to analyze replication fork dynamics in vertebrate cells rely on measuring the overall rate of DNA synthesis in a population of pulse-labeled cells. This is a quantitative approach and does not allow for qualitative analysis of parameters that influence DNA synthesis. In contrast, the rate of movement of active forks can be followed directly when using the DNA fiber technique2-4. In this approach, nascent DNA is labeled in vivo by incorporation of halogenated nucleotides (Fig 1A). Subsequently, individual fibers are stretched onto a microscope slide, and the labeled DNA replication tracts are stained with specific antibodies and visualized by fluorescence microscopy (Fig 1B). Initiation of replication as well as fork directionality is determined by the consecutive use of two differently modified analogues. Furthermore, the dual-labeling approach allows for quantitative analysis of parameters that influence DNA synthesis during the S-phase, i.e. replication structures such as ongoing and stalled forks, replication origin density as well as fork terminations. Finally, the experimental procedure can be accomplished within a day, and requires only general laboratory equipment and a fluorescence microscope.

DT40, a model vertebrate genetic system, provides a powerful tool to analyze protein function. Here we describe a simple method that allows qualitative analysis of parameters that influence DNA synthesis during the S-phase in DT40 cells at the single molecule level.

Maintenance of replication fork stability is of utmost importance for dividing cells to preserve viability and prevent disease. The processes involved not ...

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the function and regulation of protein folding was studied in vitro, in isolated tissue culture cells and in unicellular organisms. Recent studies have uncovered links between protein homeostasis (proteostasis), metabolism, development, aging, and temperature-sensing. These findings have led to the development of new tools for monitoring protein folding in the model metazoan organism Caenorhabditis elegans. In our laboratory, we combine behavioral assays, imaging and biochemical approaches using temperature-sensitive or naturally occurring metastable proteins as sensors of the folding environment to monitor protein misfolding. Behavioral assays that are associated with the misfolding of a specific protein provide a simple and powerful readout for protein folding, allowing for the fast screening of genes and conditions that modulate folding. Likewise, such misfolding can be associated with protein mislocalization in the cell. Monitoring protein localization can, therefore, highlight changes in cellular folding capacity occurring in different tissues, at various stages of development and in the face of changing conditions. Finally, using biochemical tools ex vivo, we can directly monitor protein stability and conformation. Thus, by combining behavioral assays, imaging and biochemical techniques, we are able to monitor protein misfolding at the resolution of the organism, the cell, and the protein, respectively.

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

To study the relationship between protein homeostasis, stress and aging, we monitored changes in protein folding by following protein dysfunction, protein localization in the cell and protein stability at the organismal, cellular and protein levels, using the genetically tractable metazoan Caenorhabditis elegans as a model system.

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the ...

Real-time Imaging of Single Engineered RNA Transcripts in Living Cells Using Ratiometric Bimolecular Beacons

The growing realization that both the temporal and spatial regulation of gene expression can have important consequences on cell function has led to the development of diverse techniques to visualize individual RNA transcripts in single living cells. One promising technique that has recently been described utilizes an oligonucleotide-based optical probe, ratiometric bimolecular beacon (RBMB), to detect RNA transcripts that were engineered to contain at least four tandem repeats of the RBMB target sequence in the 3&#8217;-untranslated region. RBMBs are specifically designed to emit a bright fluorescent signal upon hybridization to complementary RNA, but otherwise remain quenched. The use of a synthetic probe in this approach allows photostable, red-shifted, and highly emissive organic dyes to be used for imaging. Binding of multiple RBMBs to the engineered RNA transcripts results in discrete fluorescence spots when viewed under a wide-field fluorescent microscope. Consequently, the movement of individual RNA transcripts can be readily visualized in real-time by taking a time series of fluorescent images. Here we describe the preparation and purification of RBMBs, delivery into cells by microporation and live-cell imaging of single RNA transcripts.

Ratiometric bimolecular beacons (RBMBs) can be used to image single engineered RNA transcripts in living cells. Here, we describe the preparation and purification of RBMBs, delivery of RBMBs into cells by microporation and fluorescent imaging of single RNA transcripts in real-time.

The growing realization that both the temporal and spatial regulation of gene expression can have important consequences on cell function has led to the ...

Sampling Blood from the Lateral Tail Vein of the Rat

Blood samples are commonly obtained in many experimental contexts to measure targets of interest, including hormones, immune factors, growth factors, proteins, and glucose, yet the composition of the blood is dynamically regulated and easily perturbed. One factor that can change the blood composition is the stress response triggered by the sampling procedure, which can contribute to variability in the measures of interest. Here we describe a procedure for blood sampling from the lateral tail vein in the rat. This procedure offers significant advantages over other more commonly used techniques. It permits rapid sampling with minimal pain or invasiveness, without anesthesia or analgesia. Additionally, it can be used to obtain large volume samples (upwards of 1 ml in some rats), and it may be used repeatedly across experimental days. By minimizing the stress response and pain resulting from blood sampling, measures can more accurately reflect the true basal state of the animal, with minimal influence from the sampling procedure itself.

Blood samples are useful for assessing biomarkers of physiological states or disease in vivo. Here we describe the methodology to sample blood from the lateral tail vein in the rat. This method provides rapid samples with minimal pain and invasiveness.

Blood samples are commonly obtained in many experimental contexts to measure targets of interest, including hormones, immune factors, growth factors, ...

Genome-wide Mapping of Drug-DNA Interactions in Cells with COSMIC (Crosslinking of Small Molecules to Isolate Chromatin)

The genome is the target of some of the most effective chemotherapeutics, but most of these drugs lack DNA sequence specificity, which leads to dose-limiting toxicity and many adverse side effects. Targeting the genome with sequence-specific small molecules may enable molecules with increased therapeutic index and fewer off-target effects. N-methylpyrrole/N-methylimidazole polyamides are molecules that can be rationally designed to target specific DNA sequences with exquisite precision. And unlike most natural transcription factors, polyamides can bind to methylated and chromatinized DNA without a loss in affinity. The sequence specificity of polyamides has been extensively studied in vitro with cognate site identification (CSI) and with traditional biochemical and biophysical approaches, but the study of polyamide binding to genomic targets in cells remains elusive. Here we report a method, the crosslinking of small molecules to isolate chromatin (COSMIC), that identifies polyamide binding sites across the genome. COSMIC is similar to chromatin immunoprecipitation (ChIP), but differs in two important ways: (1) a photocrosslinker is employed to enable selective, temporally-controlled capture of polyamide binding events, and (2) the biotin affinity handle is used to purify polyamide&#8211;DNA conjugates under semi-denaturing conditions to decrease DNA that is non-covalently bound. COSMIC is a general strategy that can be used to reveal the genome-wide binding events of polyamides and other genome-targeting chemotherapeutic agents.

Genome-wide Mapping of Drug-DNA Interactions in Cells with COSMIC Crosslinking of Small Molecules to Isolate Chromatin

Identifying the direct targets of genome-targeting molecules remains a major challenge. To understand how DNA-binding molecules engage the genome, we developed a method that relies on crosslinking of small molecules to isolate chromatin (COSMIC).

The genome is the target of some of the most effective chemotherapeutics, but most of these drugs lack DNA sequence specificity, which leads to ...

Amplification, Next-generation Sequencing, and Genomic DNA Mapping of Retroviral Integration Sites

Retroviruses exhibit signature integration preferences on both the local and global scales. Here, we present a detailed protocol for (1) generation of diverse libraries of retroviral integration sites using ligation-mediated PCR (LM-PCR) amplification and next-generation sequencing (NGS), (2) mapping the genomic location of each virus-host junction using BEDTools, and (3) analyzing the data for statistical relevance. Genomic DNA extracted from infected cells is fragmented by digestion with restriction enzymes or by sonication. After suitable DNA end-repair, double-stranded linkers are ligated onto the DNA ends, and semi-nested PCR is conducted using primers complementary to both the long terminal repeat (LTR) end of the virus and the ligated linker DNA. The PCR primers carry sequences required for DNA clustering during NGS, negating the requirement for separate adapter ligation. Quality control (QC) is conducted to assess DNA fragment size distribution and adapter DNA incorporation prior to NGS. Sequence output files are filtered for LTR-containing reads, and the sequences defining the LTR and the linker are cropped away. Trimmed host cell sequences are mapped to a reference genome using BLAT and are filtered for minimally 97% identity to a unique point in the reference genome. Unique integration sites are scrutinized for adjacent nucleotide (nt) sequence and distribution relative to various genomic features. Using this protocol, integration site libraries of high complexity can be constructed from genomic DNA in three days. The entire protocol that encompasses exogenous viral infection of susceptible tissue culture cells to integration site analysis can therefore be conducted in approximately one to two weeks. Recent applications of this technology pertain to longitudinal analysis of integration sites from HIV-infected patients.

We describe a protocol for amplifying retroviral integration sites from the genomic DNA of infected cells, sequencing the amplified virus-host junctions, and then mapping these sequences to a reference genome. We also describe techniques to quantify the distribution of integration sites relative to various genomic annotations using BEDTools.

Retroviruses exhibit signature integration preferences on both the local and global scales. Here, we present a detailed protocol for (1) generation of ...