We would like to acknowledge Silvia Marchet and Constanza Lamperti (Istituto Neurologico &#34;Carlo Besta&#34;, Fondazione IRCCS, Milan) for preparing and providing the m.3243A&#62;G cybrid cells used as illustrative examples for this protocol. We would also like to acknowledge the members of the Mitochondrial Genetics Group (MRC-MBU, University of Cambridge) for useful discussion during the course of this research. This work was supported by core funding from the Medical Research Council UK (MC_UU_00015/4 and MC_UU_00028/3). P.A.N. and P.S.-P. are additionally supported by The Lily Foundation and The Champ Foundation, respectively.

Informed consent was provided for the use of the human 3243A&#62;G cybrid cells and the immortalized m.5024C&#62;T MEFs used in this study. Ethical approval was not required in this instance as the patient cells were not collected at the University of Cambridge. The use of human fibroblasts may, however, require ethical approval. It is highly recommended to follow best practices for PCR setup when preparing the sample DNA for pyrosequencing. Frequent amplification using identical primers can lead to amplicon contamination and introduce bias to the subsequent genotyping if strict separation between the pre-PCR and post-PCR areas is not observed. The pipeline presented here uses specific equipment from a sole manufacturer; the details can be found in the Table of Materials. The primer design for the PCR can be performed manually if so desired; however, it is recommended to use existing software for this purpose (see the Table of Materials).
1. Pyrosequencing primer design and assay selection
<ol>
	<li>Obtaining primer candidates with software
	<ol>
		<li>Obtain an mtDNA sequence file for the species being genotyped, and identify the position of the SNP. Ensure that the reference sequence used employs the appropriate base numbering for the mutation studied so that the position of the SNP can be easily identified.</li>
		<li>Copy 1,000 base pairs upstream and downstream of the SNP site, and paste the truncated sequence into the software intended for the pyrosequencing primer design (see the Table of Materials).</li>
		<li>Set the analyzed SNP base as the target of the pyrosequencing assay in the software by highlighting the polymorphic base and right-clicking on and then selecting set target region.</li>
		<li>Press the play icon in the top right-hand corner of the interface to launch primer selection.</li>
		<li>Wait for the software to now automatically generate primer trios: two primers for preamplification of the template DNA and a third primer for sequencing by synthesis in the pyrosequencing machine. Retain all the primer sets by right-clicking on and selecting copy all primer sets.</li>
	</ol>
	</li>
	<li>Selecting the optimal primer sets from the generated list
	<ol>
		<li>Retain the primer sets with the highest quality score based on the software output, which provides primer sets in descending order based on the quality score. If possible, omit primer sets with a score below 80.</li>
		<li>For the retained primer sets, identify and copy the amplicon produced by the amplification primers.</li>
		<li>Align the amplicons produced with the entire genome of the organism to be genotyped using NCBI Blast by using the online submission portal at&#160;https://blast.ncbi.nlm.nih.gov/Blast.cgi. From the dropdown menus during submission, select the following:
		<ol>
			<li>Select Database | Genomic + Transcript databases.</li>
			<li>Select Human or Mouse in the dropdown menu, depending on the SNP analyzed.</li>
			<li>Select Optimize for | Somewhat similar sequences (Blastn).</li>
		</ol>
		</li>
		<li>(Optional) If genotyping an organism other than a mouse or human, select the following during submission:
		<ol>
			<li>Select Database | Standard database.</li>
			<li>Select RefSeq Representative genomes in the dropdown menu</li>
			<li>Select Optimize for | Somewhat similar sequences (Blastn)</li>
		</ol>
		</li>
		<li>When possible, omit any amplicons that have perfect homology to the mtDNA amplicon, particularly if the primer binding regions are perfectly homologous (see the discussion). 
		NOTE: The BLAST alignment tool will return any sequence with considerable homology to the pre-amplification amplicons. This allows for the detection of the NUMTs described in the introduction, which if not accounted for, can induce a bias as they are co-amplified with the mitochondrial DNA.</li>
		<li>Order the oligos obtained via this pipeline. Make sure that 5&#39; biotin modification is added to the correct amplification primer during the synthesis order, such that the sequencing primer will be complementary to the biotinylated strand. 
		&#8203;NOTE: The choice of sequencing primer is more flexible than the amplification primers, and it is recommended to have at least one base separating the 3&#39; end of the sequencing primer and the variable position.</li>
	</ol>
	</li>
</ol>
2. Preamplification PCR optimization
<ol>
	<li>Prepare the DNA samples by extracting the total genomic DNA using an appropriate method. 
	NOTE: This will largely depend on the number of cells being genotyped and their origin. If isolating DNA from single cells using a lysis buffer containing proteinase K, the sample must be denatured at 95 &#176;C for 10 min so as not to disrupt the polymerase during the subsequent PCR.</li>
	<li>PCR setup and thermal block settings
	<ol>
		<li>Set up the PCR with a high-fidelity polymerase of choice. Prepare a Master Mix for four reactions. As only 10 &#181;L of PCR reaction are required for a pyrosequencing run, prepare 25 &#181;L of PCR reactions (to allow for a technical repeat if necessary). Use 40 cycles of PCR with approximately 10 ng of genomic DNA as the starting material with 1 &#181;M of both the forward and reverse primers.</li>
		<li>Set the extension time according to the manufacturer&#39;s specifications for the polymerase used, taking into consideration the length of the chosen preamplification primers.</li>
		<li>Use a thermal cycler with variable annealing temperature settings. As the annealing temperature can differ depending on the primer sequence, the salt content of the polymerase buffer, and other factors, program four different annealing temperatures into the thermal cycling program. 
		NOTE: The worked example in the representative results used the following temperatures: 55 &#176;C, 60 &#176;C, 65 &#176;C, and 70 &#176;C.</li>
		<li>Split the Master Mix into four 200 &#181;L PCR tubes, and set them to run at each of the four selected annealing temperatures in the thermal cycler for 40 cycles.</li>
	</ol>
	</li>
	<li>Preamplification band visualization
	<ol>
		<li>During the PCR run, prepare a 2% (w/v) agarose gel in 1x TBE with SYBR Safe or ethidium bromide as a visualizing agent. 
		NOTE: A higher percentage gel is recommended for visualization as the amplified fragments are usually short, typically ranging from 100 to 500 base pairs.</li>
		<li>After the PCR in step 2.2 is complete, mix 10 &#181;L of the reaction with an appropriate amount of DNA loading buffer, and run on the 2% agarose gel at 7 V/cm for approximately 45 min.</li>
		<li>Visualize the resulting DNA fragments on a UV transilluminator. 
		&#8203;NOTE: The thermal cycling conditions selected for the preamplification step should produce a single clean band of the expected size. Lower annealing temperatures can occasionally result in off-target amplification, which can introduce biases to the subsequent pyrosequencing.</li>
		<li>Select the lowest annealing temperature that produces a clean band of the correct size for subsequent amplifications.</li>
		<li>(Optional) Excise a band of the expected size, purify using a gel extraction kit of choice, and analyze by Sanger sequencing to confirm the approximate heteroplasmy of the sample as a reference.</li>
	</ol>
	</li>
</ol>
3. Instrument setup and run
NOTE: Once the PCR step in the previous section is optimized, the next step involves programming the pyrosequencer with the correct nucleotide sequence to analyze for the specific SNP. This involves entering 10 bases directly downstream of the 3&#39; end of the sequencing primer. This is detailed in the following section.
<ol>
	<li>Assay configuration
	<ol>
		<li>Open the run software provided with the pyrosequencer, and select New Assay in the top-left corner of the interface.</li>
		<li>Select the Allele quantification assay template.</li>
		<li>Input the Sequence to be analyzed into the corresponding box by typing in the nucleotides to be incorporated directly downstream of the sequencing primer, which should be upstream of the SNP of interest. For the variable position, denote the two possible bases separated by a slash (e.g., A/T).</li>
		<li>Press Generate dispensation order, and let the software automatically determine a suitable order for the nucleotides to be dispensed in the sequencing-by-synthesis reaction.</li>
		<li>Save and provide a name for the assay.</li>
	</ol>
	</li>
	<li>Reagent preparation and storage
	<ol>
		<li>Dilute the sequencing primer ordered to 4 &#181;M in the Annealing Buffer provided in pyrosequencer reagent kit. 
		NOTE: The sequencing primer can first be diluted in water to 100 &#181;M as a stock solution and subsequently further diluted in Annealing Buffer to 4 &#181;M as needed.</li>
		<li>Enzyme and substrate preparation and handling
		<ol>
			<li>When first unboxing, redissolve the lyophilized enzyme and substrate as per the manufacturer&#39;s recommendations. Store at &#8722;20 &#176;C when not in use.</li>
			<li>Upon subsequent use, thaw the enzyme and substrate vials from &#8722;20 &#176;C. 
			NOTE: All the other reagents in the provided kit can be kept refrigerated at 4 &#176;C. Diluted sequencing primers can also be kept at 4 &#176;C.</li>
		</ol>
		</li>
		<li>Place the required amount of each reagent on ice.</li>
		<li>Keep the remaining components required, namely the streptavidin-coated magnetic beads, absorber strips, and pyrosequencing disks, at 4 &#176;C.</li>
	</ol>
	</li>
	<li>Run execution 
	NOTE: When an assay is first designed, it must be calibrated using PCR products of known heteroplasmy, which ensures that the assay can accurately distinguish heteroplasmies. Researchers can use mixtures of PCR products of known heteroplasmy as standards. Samples can also be verified by other methods mentioned in the introduction and the discussion, notably NGS.
	<ol>
		<li>Dilute the DNA to be analyzed to 5 ng/&#181;L. Particularly in the first run, be sure to include samples of known heteroplasmy as a reference and/or wild-type samples.</li>
		<li>Perform a presequencing PCR of 5 &#181;L of diluted DNA in 25 &#181;L reactions using the amplification primers and parameters identified in section 1 and section 2. Perform technical PCR replicates for each sample. 
		NOTE: The preamplification PCR can be stored short term at 4 &#176;C or long term at &#8722;20 &#176;C before proceeding to sequencing.</li>
		<li>Run file setup
		<ol>
			<li>Select New Run in the top-left corner of pyrosequencer software.</li>
			<li>The pyrosequencer can simultaneously sequence 48 separate preamplified reactions, with an empty square representing a single sequencing well on a pyrosequencing disk. Load the assays configured in section 3.1 by right-clicking on a square and selecting load assay. If required, sequence up to four separate assays with different sequencing primers.</li>
			<li>Set the primer dispensation mode to Automatic to automatically assign an injection chamber for each sequencing primer used for the run.</li>
			<li>Set Run mode to Standard unless running four different types of assays on one sequencing disk.</li>
			<li>Ensure the number of assays on the run template file matches the number of PCR reactions being amplified. 
			NOTE: If running more than 48 samples, additional run files will need the be set up.</li>
			<li>Save the run files to a USB drive.</li>
		</ol>
		</li>
		<li>Priming the pyrosequencer
		<ol>
			<li>Press the Cleaning button on the main touch screen of the device, and clean all injectors with high-purity water following the instructions on the screen. When inserting the absorber strip into the machine, ensure that the ends meet in the &#34;9 o&#39;clock&#34; position (directly left of the center).</li>
			<li>Plug in the USB stick with the runs set up in step 3.3.3, and load the run files defined in step 3.3.3. 
			NOTE: The run files must be saved outside any directory or folder on the USB or they cannot be read by the machine.</li>
			<li>Follow the instructions on the device to load and prime the reagents as required into the corresponding injectors on the machine.</li>
		</ol>
		</li>
		<li>Sample disk preparation and assay launch
		<ol>
			<li>Allow the streptavidin-coated magnetic beads to equilibrate to room temperature. 
			NOTE: These beads will enable the device to capture the biotinylated strand from the preamplification PCR step. They need to be purchased separately from the kit.</li>
			<li>Load 3 &#181;L of beads into the wells defined in the run file from step 3.3.3.</li>
			<li>Load 10 &#181;L of each PCR reaction to be sequenced into the corresponding sample, and pipette up and down to mix the sample with the magnetic beads. Avoid bubbles if possible.</li>
			<li>Look for the indication in the primed pyrosequencer that the disk can be loaded into the machine. Unscrew the plate holding nut before aligning the loaded sample plate with the metal pin in the disk compartment.</li>
			<li>Once the plate is firmly screwed into the plate compartment, launch the sequencing run by pressing the start button on the touchscreen interface.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
4. Result acquisition
<ol>
	<li>Once the run is complete, remove the USB drive from the machine, and plug it back into the computer running the pyrosequencer software. While proceeding with the following steps, proceed with cleaning the pyrosequencer following the prompts if it is the final run of the day.</li>
	<li>Wait for the newly generated run file to appear on the USB drive, and double-click on the run result file. This automatically analyzes the luciferase output of each well upon nucleotide incorporation and quantifies the mitochondrial allele of interest defined during the assay configuration in section 3.1.</li>
	<li>Look for the following color scores shown by the software based on the quality of the reads. Blue indicates an optimal run, yellow a run with a warning, and red a failed run. To save the results, select Reports in the top window | Full report to generate a .pdf file containing the pyrograms and results from each well of the run. Alternatively, download the results in other formats under the Reports tab.</li>
</ol>

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M.M. is a co-founder, shareholder, and member of the Scientific Advisory Board of Pretzel Therapeutics, Inc. P.S.-P. and P.A.N. provide consultancy services for Pretzel Therapeutics, Inc.

A critical aspect for the success of the protocol is avoiding contaminations, particularly when using low amounts of starting material. It is recommended to use a UV hood and filtered pipette tips when preparing the samples wherever possible, as well as to keep the preamplification and post-amplification areas separate. Blank measurements and samples of known heteroplasmy (such as wild-type DNA) should always be included to be used as benchmarks to check for technical or biological bias. 
A no...

The mitochondrial genome exists in the form of small (16.5 kb) circular molecules (mtDNA) present in the innermost compartment of the mitochondria named the matrix and encodes 13 subunits of the mitochondrial respiratory chain, as well as the tRNAs and rRNAs necessary for their translation in situ by the mitochondrial ribosome1. This genome represents approximately 1% of all the proteins necessary for mitochondrial function, the remainder of which are encoded by the nuclear DNA (nDNA). It is commonly assumed that mitochondria are derived from an endosymbiotic fusion event between an alpha-proteobacterial ancestor and an ancestral eukaryotic cell. Once this hypothetical symbiosis took place, the genetic information of the mitochondria was gradually transferred to the nucleus over eons, which explains the aforementioned compactness of the mtDNA when compared to the genomes of modern cyanobacteria2. Such a transfer of genes is most strongly evidenced by the existence of long stretches of nDNA that are highly homologous to the sequences found in mtDNA. These nuclear mitochondrial sequences (NUMTs) are a common source of misinterpretation during genotyping, and certain precautions must be taken to avoid nuclear biases when genotyping mtDNA3 (Figure 1A).
Another distinctive feature of mtDNA is that its copy number varies depending on the cell type, numbering anywhere from tens to thousands of copies per cell4. Owing to this multi-copy nature, mtDNA can harbor a wide range of genotypes within a single cell, which can result in a more continuous distribution of alleles in contrast to the discrete alleles associated with nuclear genes when considering the zygosity of chromosomes. This heterogeneity of mitochondrial alleles is referred to as mitochondrial heteroplasmy, which is typically expressed in the percent prevalence of a given mutation as a proportion of the total mtDNA in a given cell. Heteroplasmy can be contrasted with homoplasmy, which refers to a unique species of mtDNA being present across a cell.
Measuring mitochondrial heteroplasmy is of particular interest when quantifying the proportion of mtDNA molecules harboring pathogenic variants. Such variants come in the form of single nucleotide polymorphisms (SNPs), small indels, or large-scale deletions5. Most humans are heteroplasmic for pathogenic variants; however, they do not exhibit any clinical phenotypes, which often only manifest at higher heteroplasmy levels of pathogenic mtDNA in a phenomenon referred to as the threshold effect6. While the values associated with pathogenicity are highly dependent on the nature of the pathogenic mutation and the tissue in which it occurs, they typically lie above 60% heteroplasmy7.
There are several research areas in which mitochondrial genotyping is common. In the medical field, testing for or quantifying mtDNA mutations can serve as a diagnostic criterion for mitochondrial diseases, many of which have mtDNA aberrations as their origin5. In addition to the study of human pathogenic mutations, the prevalence of animal models harboring pathogenic SNPs in the mtDNA is likely to increase, given the recent advent of mitochondrial base editing enabled by mitochondrially targeted DddA-derived cytosine base editors (DdCBEs)8 and TALE-based deaminases (TALEDs) for adenine base editing9. This approach will be instrumental in understanding the interplay between aberrant mitochondrial genotypes and the resulting dysfunctions. There is also ongoing scientific research into remodeling the mitochondrial genome for ultimate use as a therapeutic strategy in human mitochondrial diseases via an approach known as heteroplasmy shifting. This field of research primarily involves directing mutation-specific nucleases to the mitochondrial matrix; this results in the preferential degradation of pathogenic mtDNA, leading to rescues in phenotype10,11,12,13. Any experiments involving the remodeling of the mitochondrial genotype require a robust quantitative method to assess heteroplasmy shifts.
A wide variety of methods are used to genotype mtDNA, and these vary according to the nature of the mutation. Next-generation sequencing (NGS) methods are more precise when it comes to quantifying SNPs in mtDNA; however, these methods remain prohibitively expensive for the routine quantification of mitochondrial heteroplasmy, particularly if the number of samples is small. Sanger sequencing can also allow for the detection of SNPs; however, this approach is not quantitative and often fails to detect low levels of heteroplasmy or can be inaccurate when estimating high heteroplasmies. Pyrosequencing, as an assay that involves minimal preparation and enables the rapid quantification of heteroplasmy for any mtDNA sample, is proposed as an apt compromise between these two extremes. This method has been used routinely to quantify mitochondrial SNPs by numerous researchers in various contexts, including forensic analysis14,15, clinical diagnosis16, or the genotyping of mtDNA from single cells17.
This assay involves a first PCR preamplification step of a region flanking the SNP in the mtDNA, which is followed by a sequencing-by-synthesis assay using one strand of the previously generated amplicon. One of the two primers used in the preamplification step must be biotinylated on the 5&#39; end, which will enable the pyrosequencing apparatus to isolate the single strand of DNA to be used as template for the sequencing reaction. A third sequencing primer is then annealed onto the retained biotinylated strand, which allows for nascent DNA synthesis as deoxynucleotides to be dispensed in a predefined order into the reaction chamber. The pyrosequencer records the amount of each base incorporated based on a luminescent readout, allowing the relative quantification of mutant and wild-type mitochondrial alleles upon DNA synthesis (Figure 1B). The luminescence is generated by a luciferase enzyme, which emits light in the presence of ATP that an ATP sulfurylase synthesizes de novo at each incorporation event from the pyrophosphates released by each nucleotide. These two reactions can be summarized as follows:
1. PPi (from nucleotide incorporation) + APS &#8594; ATP + sulphate (ATP sulfurylase)
2. ATP + luciferin + O2 &#8594; AMP + PPi + oxyluciferin + CO2 + light (luciferase)
Detecting adenine bases by the pyrosequencer without ATP cross-reacting with the luciferase in the second reaction is a challenge. However, this is solved by using an adenine analog for DNA synthesis, namely dATP&#945;S. Despite not being a perfect substrate for luciferase, it produces a stronger luminescence compared with the three other nucleotides, which is digitally adjusted by the pyrosequencer and set to a factor of 0.9. Due to this inherent variability, it is suggested to avoid sequencing adenine at the SNP position (see the discussion for further details).
The following protocol details the method of mtDNA heteroplasmy assessment by pyrosequencing and outlines the necessary precautions in designing the amplification primers to avoid biological or technical bias when genotyping SNPs in mtDNA. The latter involves digitally surveying and selecting the primer sets, optimizing the preamplification PCR, and finally, sequencing and refining the assay. Two applied example assays are demonstrated: first, the optimization of the most common human pathogenic variant m.3243A&#62;G18, and second, the genotyping of mouse embryonic fibroblast (MEF) cells that have undergone heteroplasmy shifting using technologies developed at the Minczuk laboratory in Cambridge10,11,12,19,20,21,22.

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genotyping single nucleotide polymorphisms in the mitochondrial genome by pyrosequencing

Mutations in the mitochondrial genome (mtDNA) have been associated with maternally inherited genetic diseases. However, interest in mtDNA polymorphisms has increased in recent years due to the recently developed ability to produce models by mtDNA mutagenesis and a new appreciation of the association between mitochondrial genetic aberrations and common age-related diseases such as cancer, diabetes, and dementia. Pyrosequencing is a sequencing-by-synthesis technique that is widely employed across the mitochondrial field for routine genotyping experiments. Its relative affordability when compared to massive parallel sequencing methods and ease of implementation make it an invaluable technique in the field of mitochondrial genetics, allowing for the rapid quantification of heteroplasmy with increased flexibility. Despite the practicality of this method, its implementation as a means of mtDNA genotyping requires the observation of certain guidelines, specifically to avoid certain biases of biological or technical origin. This protocol outlines the necessary steps and precautions in designing and implementing pyrosequencing assays for use in the context of heteroplasmy measurement.

This section presents an example optimization of a pyrosequencing assay for a human pathogenic mtDNA mutation, as well as sequencing data from the genotyping of heteroplasmic (m.5024C&#62;T) mouse embryonic fibroblasts (MEFs) treated with mitochondrial zinc finger nucleases (mtZFNs). Optimizing the assay for human cells and comparing two different assays demonstrates how to select the most accurate one, whereas genotyping genetically modified MEF cells in the second example serves as an applied example of detecting heter...

Watch this Scientific Journal Video about Genotyping Single Nucleotide Polymorphisms in the Mitochondrial Genome by Pyrosequencing at JoVE.com

Genotyping Single Nucleotide Polymorphisms in the Mitochondrial Genome by Pyrosequencing

Pyrosequencing assays enable the robust and rapid genotyping of mitochondrial DNA single nucleotide polymorphisms in heteroplasmic cells or tissues.

Mutations in the mitochondrial genome (mtDNA) have been associated with maternally inherited genetic diseases. However, interest in mtDNA polymorphisms ...

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1.3K Views.  University of Cambridge, Cambridge Biomedical Campus. Pyrosequencing assays enable the robust and rapid genotyping of mitochondrial DNA single nucleotide polymorphisms in heteroplasmic cells or tissues. 

Video: Genotyping Single Nucleotide Polymorphisms in the Mitochondrial Genome by Pyrosequencing

The Use of Primary Human Fibroblasts for Monitoring Mitochondrial Phenotypes in the Field of Parkinson's Disease

Parkinson's disease (PD) is the second most common movement disorder and affects 1% of people over the age of 60 1. Because ageing is the most important risk factor, cases of PD will increase during the next decades 2. Next to pathological protein folding and impaired protein degradation pathways, alterations of mitochondrial function and morphology were pointed out as further hallmark of neurodegeneration in PD 3-11.
After years of research in murine and human cancer cells as in vitro models to dissect molecular pathways of Parkinsonism, the use of human fibroblasts from patients and appropriate controls as ex vivo models has become a valuable research tool, if potential caveats are considered. Other than immortalized, rather artificial cell models, primary fibroblasts from patients carrying disease-associated mutations apparently reflect important pathological features of the human disease.
Here we delineate the procedure of taking skin biopsies, culturing human fibroblasts and using detailed protocols for essential microscopic techniques to define mitochondrial phenotypes. These were used to investigate different features associated with PD that are relevant to mitochondrial function and dynamics. Ex vivo, mitochondria can be analyzed in terms of their function, morphology, colocalization with lysosomes (the organelles degrading dysfunctional mitochondria) and degradation via the lysosomal pathway. These phenotypes are highly relevant for the identification of early signs of PD and may precede clinical motor symptoms in human disease-gene carriers. Hence, the assays presented here can be utilized as valuable tools to identify pathological features of neurodegeneration and help to define new therapeutic strategies in PD.

Fibroblasts from patients carrying mutations in Parkinson's disease-causing genes represent an easily accessible ex vivo model to study disease-associated phenotypes. Live cell imaging gives the opportunity to study morphological and functional parameters in living cells. Here we describe the preparation of human fibroblasts and subsequent monitoring of mitochondrial phenotypes.

Parkinson's disease (PD) is the second most  common movement disorder and affects 1% of people over the age of 60 1. Because ageing is  the most important ...

Identification of Sleeping Beauty Transposon Insertions in Solid Tumors using Linker-mediated PCR

Genomic, proteomic, transcriptomic, and epigenomic analyses of human tumors indicate that there are thousands of anomalies within each cancer genome compared to matched normal tissue. Based on these analyses it is evident that there are many undiscovered genetic drivers of cancer1. Unfortunately these drivers are hidden within a much larger number of passenger anomalies in the genome that do not directly contribute to tumor formation. Another aspect of the cancer genome is that there is considerable genetic heterogeneity within similar tumor types. Each tumor can harbor different mutations that provide a selective advantage for tumor formation2. Performing an unbiased forward genetic screen in mice provides the tools to generate tumors and analyze their genetic composition, while reducing the background of passenger mutations. The Sleeping Beauty (SB) transposon system is one such method3. The SB system utilizes mobile vectors (transposons) that can be inserted throughout the genome by the transposase enzyme. Mutations are limited to a specific cell type through the use of a conditional transposase allele that is activated by Cre Recombinase. Many mouse lines exist that express Cre Recombinase in specific tissues. By crossing one of these lines to the conditional transposase allele (e.g. Lox-stop-Lox-SB11), the SB system is activated only in cells that express Cre Recombinase. The Cre Recombinase will excise a stop cassette that blocks expression of the transposase allele, thereby activating transposon mutagenesis within the designated cell type. An SB screen is initiated by breeding three strains of transgenic mice so that the experimental mice carry a conditional transposase allele, a concatamer of transposons, and a tissue-specific Cre Recombinase allele. These mice are allowed to age until tumors form and they become moribund. The mice are then necropsied and genomic DNA is isolated from the tumors. Next, the genomic DNA is subjected to linker-mediated-PCR (LM-PCR) that results in amplification of genomic loci containing an SB transposon. LM-PCR performed on a single tumor will result in hundreds of distinct amplicons representing the hundreds of genomic loci containing transposon insertions in a single tumor4. The transposon insertions in all tumors are analyzed and common insertion sites (CISs) are identified using an appropriate statistical method5. Genes within the CIS are highly likely to be oncogenes or tumor suppressor genes, and are considered candidate cancer genes. The advantages of using the SB system to identify candidate cancer genes are: 1) the transposon can easily be located in the genome because its sequence is known, 2) transposition can be directed to almost any cell type and 3) the transposon is capable of introducing both gain- and loss-of-function mutations6. The following protocol describes how to devise and execute a forward genetic screen using the SB transposon system to identify candidate cancer genes (Figure 1).

Identification of Sleeping Beauty Transposon Insertions in Solid Tumors using Linker-mediated PCR

A method of identifying unknown drivers of carcinogenesis using an unbiased approach is described. The method uses the Sleeping Beauty transposon as a random mutagen directed to specific tissues. Genomic mapping of transposon insertions that drive tumor formation identifies novel oncogenes and tumor suppressor genes

Genomic, proteomic, transcriptomic, and epigenomic analyses of human tumors indicate that there are thousands of anomalies within each cancer genome ...

Collection and Extraction of Saliva DNA for Next Generation Sequencing

The preferred source of DNA in human genetics research is blood, or cell lines derived from blood, as these sources yield large quantities of high quality DNA. However, DNA extraction from saliva can yield high quality DNA with little to no degradation/fragmentation that is suitable for a variety of DNA assays without the expense of a phlebotomist and can even be acquired through the mail. However, at present, no saliva DNA collection/extraction protocols for next generation sequencing have been presented in the literature. This protocol optimizes parameters of saliva collection/storage and DNA extraction to be of sufficient quality and quantity for DNA assays with the highest standards, including microarray genotyping and next generation sequencing.

DNA extraction from saliva can provide a readily available source of high molecular weight DNA, with little to no degradation/fragmentation. This protocol provides optimized parameters for saliva collection/storage and DNA extraction to be of sufficient quality and quantity for downstream DNA assays with high quality requirements.

The preferred source of DNA in human genetics research is blood, or cell lines derived from blood, as these sources yield large quantities of high quality ...

Single-stage Dynamic Reanimation of the Smile in Irreversible Facial Paralysis by Free Functional Muscle Transfer

Unilateral facial paralysis is a common disease that is associated with significant functional, aesthetic and psychological issues. Though idiopathic facial paralysis (Bell&#8217;s palsy) is the most common diagnosis, patients can also present with a history of physical trauma, infectious disease, tumor, or iatrogenic facial paralysis. Early repair within one year of injury can be achieved by direct nerve repair, cross-face nerve grafting or regional nerve transfer. It is due to muscle atrophy that in long lasting facial paralysis complex reconstructive methods have to be applied. Instead of one single procedure, different surgical approaches have to be considered to alleviate the various components of the paralysis.
The reconstruction of a spontaneous dynamic smile with a symmetric resting tone is a crucial factor to overcome the functional deficits and the social handicap that are associated with facial paralysis. Although numerous surgical techniques have been described, a two-stage approach with an initial cross-facial nerve grafting followed by a free functional muscle transfer is most frequently applied. In selected patients however, a single-stage reconstruction using the motor nerve to the masseter as donor nerve is superior to a two-stage repair. The gracilis muscle is most commonly used for reconstruction, as it presents with a constant anatomy, a simple dissection and minimal donor site morbidity.
Here we demonstrate the pre-operative work-up, the post-operative management, and precisely describe the surgical procedure of single-stage microsurgical reconstruction of the smile by free functional gracilis muscle transfer in a step by step protocol. We further illustrate common pitfalls and provide useful tips which should enable the reader to truly comprehend the procedure. We further discuss indications and limitations of the technique and demonstrate representative results.

The use of the masseteric nerve as donor nerve represents a single-stage alternative to the criterion standard two-stage procedure of cross-facial nerve grafting and free muscle transfer in facial paralysis. We provide a detailed description to safely perform this technique with a gracilis muscle transfer and discuss indications and limitations.

Unilateral facial paralysis is a common disease that is associated with significant functional, aesthetic and psychological issues. Though idiopathic ...

Rose Bengal Photothrombosis by Confocal Optical Imaging In Vivo: A Model of Single Vessel Stroke

In vivo imaging techniques have increased in utilization due to recent advances in imaging dyes and optical technologies, allowing for the ability to image cellular events in an intact animal. Additionally, the ability to induce physiological disease states such as stroke in vivo increases its utility. The technique described herein allows for physiological assessment of cellular responses within the CNS following a stroke and can be adapted for other pathological conditions being studied. The technique presented uses laser excitation of the photosensitive dye Rose Bengal in vivo to induce a focal ischemic event in a single blood vessel. 
The video protocol demonstrates the preparation of a thin-skulled cranial window over the somatosensory cortex in a mouse for the induction of a Rose Bengal photothrombotic event keeping injury to the underlying dura matter and brain at a minimum. Surgical preparation is initially performed under a dissecting microscope with a custom-made surgical/imaging platform, which is then transferred to a confocal microscope equipped with an inverted objective adaptor. Representative images acquired utilizing this protocol are presented as well as time-lapse sequences of stroke induction. This technique is powerful in that the same area can be imaged repeatedly on subsequent days facilitating longitudinal in vivo studies of pathological processes following stroke.

Rose Bengal Photothrombosis by Confocal Optical Imaging In Vivo:  A Model of Single Vessel Stroke

Here, we describe a semi-invasive optical microscopy approach for the induction of a Rose Bengal photothrombotic clot in the somatosensory cortex of a mouse in vivo. The technical aspects of the imaging procedure are described from induction of a photothrombotic event to application and data collection.

In vivo imaging techniques have increased in utilization due to recent advances in imaging dyes and optical technologies, allowing for the ability to ...

Phosphorus-31 Magnetic Resonance Spectroscopy: A Tool for Measuring In Vivo Mitochondrial Oxidative Phosphorylation Capacity in Human Skeletal Muscle

Skeletal muscle mitochondrial oxidative phosphorylation (OXPHOS) capacity, which is critically important in health and disease, can be measured in vivo and noninvasively in humans via phosphorus-31 magnetic resonance spectroscopy (31PMRS). However, the approach has not been widely adopted in translational and clinical research, with variations in methodology and limited guidance from the literature. Increased optimization, standardization, and dissemination of methods for in vivo 31PMRS would facilitate the development of targeted therapies to improve OXPHOS capacity and could ultimately favorably impact cardiovascular health. 31PMRS produces a noninvasive, in vivo measure of OXPHOS capacity in human skeletal muscle, as opposed to alternative measures obtained from explanted and potentially altered mitochondria via muscle biopsy. It relies upon only modest additional instrumentation beyond what is already in place on magnetic resonance scanners available for clinical and translational research at most institutions. In this work, we outline a method to measure in vivo skeletal muscle OXPHOS. The technique is demonstrated using a 1.5 Tesla whole-body MR scanner equipped with the suitable hardware and software for 31PMRS, and we explain a simple and robust protocol for in-magnet resistive exercise to rapidly fatigue the quadriceps muscle. Reproducibility and feasibility are demonstrated in volunteers as well as subjects over a wide range of functional capacities.

Phosphorus-31 Magnetic Resonance Spectroscopy: A Tool for Measuring In Vivo Mitochondrial Oxidative Phosphorylation Capacity in Human Skeletal Muscle

This work demonstrates the feasibility of an in vivo phosphorus-31 magnetic resonance spectroscopy (31PMRS) technique to quantify mitochondrial oxidative phosphorylation (OXPHOS) capacity in human skeletal muscle.

Skeletal muscle mitochondrial oxidative phosphorylation (OXPHOS) capacity, which is critically important in health and disease, can be measured in vivo ...

An Ex Vivo Tissue Culture Model for Fibrovascular Complications in Proliferative Diabetic Retinopathy

Diabetic retinopathy (DR) is the most common microvascular complication of diabetes and one of the leading causes of blindness in working-age adults. No current animal models of diabetes and oxygen-induced retinopathy develop the full-range progressive changes manifested in human proliferative diabetic retinopathy (PDR). Therefore, understanding of the disease pathogenesis and pathophysiology has relied largely on the use of histological sections and vitreous samples in approaches that only provide steady-state information on the involved pathogenic factors. Increasing evidence indicates that dynamic cell-cell and cell-extracellular matrix (ECM) interactions in the context of three-dimensional (3D) microenvironments are essential for the mechanistic and functional studies towards the development of new treatment strategies. Therefore, we hypothesized that the pathological fibrovascular tissue surgically excised from eyes with PDR could be utilized to reliably unravel the cellular and molecular mechanisms of this devastating disease and to test the potential for novel clinical interventions. Towards this end, we developed a novel method for 3D ex vivo culture of surgically-excised patient-derived fibrovascular tissue (FT), which will serve as a relevant model of human PDR pathophysiology. The FTs are dissected into explants and embedded in fibrin matrix for ex vivo culture and 3D characterization. Whole-mount immunofluorescence of the native FTs and end-point cultures allows thorough investigation of tissue composition and multicellular processes, highlighting the importance of 3D tissue-level characterization for uncovering relevant features of PDR pathophysiology. This model will allow the simultaneous assessment of molecular mechanisms, cellular/tissue processes and treatment responses in the complex context of dynamic biochemical and physical interactions within the PDR tissue architecture and microenvironment. Since this model recapitulates PDR pathophysiology, it will also be amenable for testing or developing new treatments.

Here, we present a protocol to study the pathophysiology of proliferative diabetic retinopathy by using patient-derived, surgically-excised, fibrovascular tissues for three-dimensional native tissue characterization and ex vivo culture. This ex vivo culture model is also amenable for testing or developing new treatments.

Diabetic retinopathy (DR) is the most common microvascular complication of diabetes and one of the leading causes of blindness in working-age adults. No ...

Organ Ischemia-Reperfusion Injury by Simulating Hemodynamic Changes in Rat Liver Transplant Model

Orthotopic liver transplantation (OLT) in rats is a tried and proven animal model used for preoperative, intraoperative, and postoperative studies, including ischemia-reperfusion injury (IRI) of extrahepatic organs. This model requires numerous experiments and devices. The duration of anhepatic phase is closely related to the time to develop IRI after transplantation. In this experiment, we used hemodynamic changes to induce extrahepatic organ damage in rats and determined the maximum tolerance time. The time until the most severe organ injury varied for different organs. This method can easily be replicated and can also be used to study IRI of the extrahepatic organs after liver transplantation.

This paper provides a detailed description of how to build an animal model of the anhepatic phase (liver ischemia) in rats to facilitate basic research into ischemia-reperfusion injury after liver transplantation.

Orthotopic liver transplantation (OLT) in rats is a tried and proven animal model used for preoperative, intraoperative, and postoperative studies, ...

Cox-Maze IV Procedure Concomitant with Valvular Surgery In Situs Inversus Dextrocardia: A Single-Center Experience in China

Atrial fibrillation (AF) is the most common cardiac arrhythmia. The use of ablation technologies made the Cox-Maze IV procedure (CMP-IV) technically easier, faster, becoming the gold standard for the surgical treatment of AF. However, the efficacy and safety of CMP-IV in situs inversus dextrocardia are largely unknown. This paper summarizes the CMP-IV procedure performed concomitantly with valvular surgery in patients with situs inversus dextrocardia at this institution.
From February 2016 to September 2020, three dextrocardia patients with persistent AF and valvular diseases were referred to this institution for valvular and CMP-IV surgery. CMP-IV was performed using either cryoablation with a nitrous oxide (N2O)-based cryoprobe or a bipolar radiofrequency clamp and bipolar radiofrequency pen. Mechanical valve replacement or mitral vavuloplasty was performed in another patient in addition to tricuspid annuloplasty. Transmurality of the ablated atrial tissues was evaluated by electron microscopy. Heart function was assessed by transthoracic echocardiography. Cardiac rhythm was monitored by 24 h Holter at 3, 6, 12, 18, 24, and 48 months follow-up. 
All the AF was successfully eliminated in the ablation procedure without recurrence or other complications during hospitalization. The mean bypass and crossclamp times were similar in all the patients. The postoperative ventilator support time, the duration of stay in the ICU, and postoperative residence time were also not significantly different among the patients. Transmural atrial necrosis was detected in the ablated atrial tissues. Sinus rhythm maintenance was achieved at 3, 6, 12, 18, 24, and 48 months follow-up in all the patients. All valve protheses switched freely; no tricuspid regurgitation was observed. The results of the present study demonstrate that the CMP-IV is safe and effective in eliminating AF in dextrocardia patients concomitant with valvular surgery.

We summarize the Cox-Maze IV procedure concomitant with valvular surgery performed in patients with situs inversus dextrocardia at this institution.

Atrial fibrillation (AF) is the most common cardiac arrhythmia. The use of ablation technologies made the Cox-Maze IV procedure (CMP-IV) technically ...

Measuring Single-Cell Mitochondrial DNA Copy Number and Heteroplasmy Using Digital Droplet Polymerase Chain Reaction

The mammalian mitochondrial (mt)DNA is a small, circular, double-stranded, intra-mitochondrial DNA molecule, encoding 13 subunits of the electron transport chain. Unlike the diploid nuclear genome, most cells contain many more copies of mtDNA, ranging from less than 100 to over 200,000 copies depending on cell type. MtDNA copy number is increasingly used as a biomarker for a number of age-related degenerative conditions and diseases, and thus, accurate measurement of the mtDNA copy number is becoming a key tool in both research and diagnostic settings. Mutations in the mtDNA, often occurring as single nucleotide polymorphisms (SNPs) or deletions, can either exist in all copies of the mtDNA within the cell (termed homoplasmy) or as a mixture of mutated and WT mtDNA copies (termed heteroplasmy). Heteroplasmic mtDNA mutations are a major cause of clinical mitochondrial pathology, either in rare diseases or in a growing number of common late-onset diseases such as Parkinson's disease. Determining the level of heteroplasmy present in cells is a critical step in the diagnosis of rare mitochondrial diseases and in research aimed at understanding common late-onset disorders where mitochondria may play a role. MtDNA copy number and heteroplasmy have traditionally been measured by quantitative (q)PCR-based assays or deep sequencing. However, the recent introduction of ddPCR technology has provided an alternative method for measuring both parameters. It offers several advantages over existing methods, including the ability to measure absolute mtDNA copy number and sufficient sensitivity to make accurate measurements from single cells even at low copy numbers. Presented here is a detailed protocol describing the measurement of mtDNA copy number in single cells using ddPCR, referred to as droplet generation PCR henceforth, with the option for simultaneous measurement of heteroplasmy in cells with mtDNA deletions. The possibility of expanding this method to measure heteroplasmy in cells with mtDNA SNPs is also discussed.

Here we present a protocol for measuring absolute mitochondrial (mt)DNA copy number and mtDNA deletion heteroplasmy levels in single cells.

Genotyping Single Nucleotide Polymorphisms in the Mitochondrial Genome by Pyrosequencing

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The Use of Primary Human Fibroblasts for Monitoring Mitochondrial Phenotypes in the Field of Parkinson's Disease

Identification of Sleeping Beauty Transposon Insertions in Solid Tumors using Linker-mediated PCR

Collection and Extraction of Saliva DNA for Next Generation Sequencing

Single-stage Dynamic Reanimation of the Smile in Irreversible Facial Paralysis by Free Functional Muscle Transfer

Rose Bengal Photothrombosis by Confocal Optical Imaging In Vivo: A Model of Single Vessel Stroke

Phosphorus-31 Magnetic Resonance Spectroscopy: A Tool for Measuring In Vivo Mitochondrial Oxidative Phosphorylation Capacity in Human Skeletal Muscle

An Ex Vivo Tissue Culture Model for Fibrovascular Complications in Proliferative Diabetic Retinopathy

Organ Ischemia-Reperfusion Injury by Simulating Hemodynamic Changes in Rat Liver Transplant Model

Cox-Maze IV Procedure Concomitant with Valvular Surgery In Situs Inversus Dextrocardia: A Single-Center Experience in China

Measuring Single-Cell Mitochondrial DNA Copy Number and Heteroplasmy Using Digital Droplet Polymerase Chain Reaction