Name: a strategy to identify de novo mutations in common disorders such as autism and schizophrenia
Uploaded: 2011-06-15T12:00:00
Duration: 5 min 51 s
Description: Watch this Scientific Journal Video about A Strategy to Identify de Novo Mutations in Common Disorders such as Autism and Schizophrenia at JoVE.com

We thanks our funding sources Genome Canada and G&eacute;nome Qu&eacute;bec, and Universit&eacute; de Montr&eacute;al as well as funding from the Canadian Foundation for Innovation for funding our 'Synapse to Disease' (S2D) project.

1. Selection of disease that may be caused by de novo mutations
A disease that corresponds to the following criteria can fit with the de novo mutation hypothesis: 
<ol>
 <li>The reproductive fitness is reduced.</li>
 <li>The frequency of the disease is relatively high and constant despite widely varying environments.</li>
 <li>The disease is associated with a higher paternal age.</li>
 <li>The classic linkage and association studies failed to explain a significant fraction of the disease heritability.</li>
 <li>The twin concordance data support a de novo model. </li>
</ol>
Analysis of the likelihood that a common disease where de novo mutations may in part explain the genetic basis is a critical first step.
2. Selection of cases and DNA samples
Selection of appropriate samples is critical for the success of the identification of de novo mutations. To maximize the chance of finding de novo mutations, we recommend the following:
<ol>
 <li>Select cases with early age of onset, severe phenotype, with unaffected parents, older fathers and with no extended family history of the disease. </li>
 <li>Choose patients whose available DNAs are sufficient to conduct the study. Especially critical is the availability of DNA from a primary cell source that was not subjected to culturing (e.g. Blood DNA or saliva DNA), </li>
 <li>The availability of both parents DNA is critical in order to determine the mutation transmission status (inherited vs. de novo). Availability of additional affected cohorts and normal controls is necessary for genetic validation studies once candidate genes are identified. </li>
 <li>Estimate the sample size based on the mutations rate, the amount of genes to be screened and the estimate of the fraction of cases that may result from a de novo mutation. </li>
</ol>
3. Gene resequencing; two major approaches
<ol>
 <li>High quality low throughput sequencing 
 This approach is based on the candidate genes approaches.
 <ol>
 	<li>Selection of candidate gene(s) 
		Select the best candidate genes based on a scoring system which is built on 6 major criteria. Then calculate the total that corresponded to the sum of all the points attributed using the six criteria listed in Table 1. See example from our project in figure 1 of selected and not selected genes distribution. 
<img src="/files/ftp_upload/2534/2534table1.jpg" alt="Table 1"/> 
 Table 1. Criteria used for the candidate gene selection 
<img src="/files/ftp_upload/2534/2534fig1.jpg" alt="Figure 1"/> 
 Figure 1. Graph showing the distribution of selected and not selected genes for sequencing in our project. We obtained a distribution of genes ranked by candidate properties by sorting genes according to their score value. For example, SHANK3 and NRXN1 genes, two genes that we found de novo mutation, had a score 7 and 6 respectively (maximum is 12).</li>
	 	<li>Design primers using Primers3 software through Exonprimer. Only coding region and splice junction should be covered including an extra 50 base pairs on each side of the exon.</li>
	 	<li>Optimize PCR conditions for the choice of Taq, reaction volume, etc.</li>
	 	<li>Optimize all PCR fragments </li>
	 	<li>Amplify 5 ng of genomic DNA extracted from blood samples according to standard procedures</li>
	 	<li>Before sending for sequencing, do quality control of your PCR products by loading a 2% agarose gel. Selected randomly samples.</li>
	 	<li>Sequence the PCR products on a DNA Analyzer on one strand. A fragment is considered successfully sequenced if the analysis of over 90% of the traces is possible. This is applicable for a large scale screening.</li>
		<li>Variants Detection
		 <ol>
		 <li>Use tools for detection and genotyping of genomic variations such as PolyPhred, Polyscan and Mutation Surveyor. A combination of more than 2 detection tools is ideal. For example, PolyPhred v.5 and PolyPhred v.6 with the default settings do not detect the same variations. Polyscan v.3 has a higher false positive mutation rate for SNPs (96%) and less for the INDELs (93%). PolyPhred v.6 did not detected the majority of true INDELs but have a false positive mutation rate (for INDELs) lower than Polyscan v.3 overall (90%). We should remove the option of SNPs detection for Polyscan v.3 and keep both PolyPhred v.5 and PolyPhred v.6 for variant detection. Mutation Surveyor and Polyscan are better for detecting indels. Note: The option of SNP detection should not be applied when using Polyscan. Only the "indel" option should be on. Polyscan generated to many false positive for SNP detection.</li>
		 <li>For each unique novel exonic variants detected, confirm it manually by reamplifying the fragment and resequencing the proband and both parents using reverse and forward primers to eliminate any technical artifact. </li>
		 </ol>
		</li>
	</ol>
 </li>
 <li>Whole exome sequencing 
 This approach is a high throughput sequencing targeting the majority of coding regions of the human genomes. We are now currently using this new approach in our lab, accelerating the detection of potential candidate genes.
		<ol>
 		<li>Order the "SureSelect Human All Exon" targeting the coding region of over 16,000 genes (50 MB of the genome) designed by Agilent or any other similar product by others like Roche . Prior to order the capture kit, the sequencing platforms should be determine (Illumina vs SOLiD).</li>
		 <li>Do the capture according to the Agilent protocol</li>
		 <li>Sequence the product on your respective available next-generation platform</li>
		 <li>Variant detection from the whole exome sequencing: Several bioinformatics tools for detection and genotyping of genomic variations from the next-generation sequencing platform are available such as BWA, Bfast, Bioscope which will perform the alignment. After which additional freely available downstream tools (for example SAM tools, Varscan, Annovar) would be needed it to call and annotate the variants. Commercial software that incorporates sequence alignment and variant calling and annotation are also available such as, NextGEN (Softgenetics), CLC Bio, and others</li>
	 </ol>
 </li>
</ol>
4. Genomic variants prioritization
Identified variants are then prioritized for follow up according to their probability in being de novo and deleterious to protein or mRNA function and /or structure. The variant follow up priorities for detection of de novo variant should be as follow:
<ol>
 <li>Unique variations (observed once in a single case)</li>
 <li>Variations not present in the parents</li>
 <li>Protein-truncating variations: nonsense, indels leading to frameshift and splicing mutations.</li>
 <li>Missense and silent variation predicted to be functionally disruptive (e.g., affect mRNA splicing). Use Polyphen, SIFT and PANTHER for functional prediction effect on the protein.</li>
</ol>
If using whole exome sequencing, selection of candidate genes can be used as a strategy for prioritizing variants for further study.
5. Genetic validation 
<ol>
 <li>Resequence the entire gene in additional patient cases (to identify other causative mutations) and in controls. The control samples will be used to evaluate allelic frequencies of prioritized variants. Any gene containing at least two different de novo deleterious mutations (nonsense, splicing, frameshifts, and predicted damaging missense) found in different patients (but not in control samples or public databases) should be highly prioritized for further validation studies. This includes: 1) testing for a potential splicing defect in lymphoblastoid cell lines derived from the patient. In our experience, most genes yield RT-PCR products from lymphoblastoid cell lines; 2) investigate altered protein expression levels by quantitative Western blot analysis of protein extracts from the lymphoblastoid cell lines and 3) further test the mutation/gene at the functional level in animal (c elegans, Zebrafish) and cell line models as previously done by our group (ex: 5,6). </li>
</ol>
6. Representative Results: 
Following this protocol, we were able to identify new genes for schizophrenia and autism. One example is our recently SHANK3 gene discovery (Figure 2). Two different de novo mutations in SHANK3 gene, one nonsense mutation found in three affected brother and one missense mutation in one affected female. 
<img src="/files/ftp_upload/2534/2534fig2.jpg" alt="Figure 2"/> 
 Figure 2. (A) Segregation of the R1117X nonsense mutation in three affected brothers of family PED 419. The proband is indicated by the arrow. (B) Segregation of the R536W missense mutation in the proband but not her non-affected brother in PED 56.

<ol>
	<li>Bassett, A. S., Bury, A., Hodgkinson, K. A., Honer, W. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reproductive+fitness+in+familial+schizophrenia.">Reproductive fitness in familial schizophrenia.</a> Schizophr Res. 21, 151-160 (1996).</li><li>Jablensky, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Schizophrenia:+manifestations,+incidence+and+course+in+different+cultures.+A+World+Health+Organization+ten-country+study.">Schizophrenia: manifestations, incidence and course in different cultures. A World Health Organization ten-country study.</a> Psychol Med Monogr. , 1-97 (1992).</li><li>Malaspina, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Schizophrenia+risk+and+paternal+age:+a+potential+role+for+de+novo+mutations+in+schizophrenia+vulnerability+genes.">Schizophrenia risk and paternal age: a potential role for de novo mutations in schizophrenia vulnerability genes.</a> CNS Spectr. 7, 26-29 (2002).</li><li>DeLisi, L. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+genome-wide+scan+for+linkage+to+chromosomal+regions+in+382+sibling+pairs+with+schizophrenia+or+schizoaffective+disorder.">A genome-wide scan for linkage to chromosomal regions in 382 sibling pairs with schizophrenia or schizoaffective disorder.</a> Am J Psychiatry. 159, 803-812 (2002).</li><li>Gauthier, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=De+Novo+SHANK3+Mutations+in+Patients+Ascertained+for+Schizophrenia.">De Novo SHANK3 Mutations in Patients Ascertained for Schizophrenia.</a> Proc Natl Acad Sci U S A. , (2010).</li><li>Piton, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mutations+in+the+calcium-related+gene+IL1RAPL1+are+associated+with+autism.">Mutations in the calcium-related gene IL1RAPL1 are associated with autism.</a> Hum Mol Genet. 17, 3965-3974 (2008).</li><li>Krawczak, M., Ball, E. V., Cooper, D. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neighboring-nucleotide+effects+on+the+rates+of+germ-line+single-base-pair+substitution+in+human+genes.">Neighboring-nucleotide effects on the rates of germ-line single-base-pair substitution in human genes.</a> Am J Hum Genet. 63, 474-488 (1998).</li></ol>

No conflicts of interest declared.

The procedure outlined here aims to identify specific common diseases that likely result, in part, from de novo mutations, and to prove this hypothesis. De novo mutations are a well established mechanism for the development of a number of diseases, for example the hereditary cancer syndromes, but has been poorly explored in common diseases. This in part results from the technical challenges involved in the identification of de novo mutations, which requires the sequencing of large amounts of DNA, which has only very recently become cost effective with the advent of Next Generation Sequencing. In addition, the de novo mutation rate in humans was, until very recently, only an estimate. Only very recently have there been reports directly determining the mutation rate in humans. Prior to these measurements, it was difficult to predict the sample size needed for this kind of study and to determine if the observed de novo mutation rate is greater than the baseline rate. Sequencing candidate genes versus whole genome? Since the majority of reported disease mutations are missense/nonsense mutations and are splice site mutations (according to HGMD web site) our screening strategy would identify over 68% of known mutations. There is also a clear relationship between the severity of amino acid replacement and the likelihood of a clinical phenotype. As compared with a conservative amino acid substitution, a nonsense change is 9.0 times more likely to present clinically 7. Thus, at this time sequencing candidate genes is the most cost effective strategy. 
The success of the outlined procedure depends on several critical steps, which are outlined in detail and illustrated using two examples, autism and schizophrenia. There are many pitfalls which need to be avoided, such as which disease to select, which patients to screen, source of DNA, and details of how to efficiently identify the de novo mutations. We provide a method for most efficiently determining the fraction of cases of any disease which results from such spontaneous mutations.

a strategy to identify de novo mutations in common disorders such as autism and schizophrenia

There are several lines of evidence supporting the role of de novo mutations as a mechanism for common disorders, such as autism and schizophrenia. First, the de novo mutation rate in humans is relatively high, so new mutations are generated at a high frequency in the population. However, de novo mutations have not been reported in most common diseases. Mutations in genes leading to severe diseases where there is a strong negative selection against the phenotype, such as lethality in embryonic stages or reduced reproductive fitness, will not be transmitted to multiple family members, and therefore will not be detected by linkage gene mapping or association studies. The observation of very high concordance in monozygotic twins and very low concordance in dizygotic twins also strongly supports the hypothesis that a significant fraction of cases may result from new mutations. Such is the case for diseases such as autism and schizophrenia. Second, despite reduced reproductive fitness1 and extremely variable environmental factors, the incidence of some diseases is maintained worldwide at a relatively high and constant rate. This is the case for autism and schizophrenia, with an incidence of approximately 1% worldwide. Mutational load can be thought of as a balance between selection for or against a deleterious mutation and its production by de novo mutation. Lower rates of reproduction constitute a negative selection factor that should reduce the number of mutant alleles in the population, ultimately leading to decreased disease prevalence. These selective pressures tend to be of different intensity in different environments. Nonetheless, these severe mental disorders have been maintained at a constant relatively high prevalence in the worldwide population across a wide range of cultures and countries despite a strong negative selection against them2. This is not what one would predict in diseases with reduced reproductive fitness, unless there was a high new mutation rate. Finally, the effects of paternal age: there is a significantly increased risk of the disease with increasing paternal age, which could result from the age related increase in paternal de novo mutations. This is the case for autism and schizophrenia3. The male-to-female ratio of mutation rate is estimated at about 4&#x2013;6:1, presumably due to a higher number of germ-cell divisions with age in males. Therefore, one would predict that de novo mutations would more frequently come from males, particularly older males4. A high rate of new mutations may in part explain why genetic studies have so far failed to identify many genes predisposing to complexes diseases genes, such as autism and schizophrenia, and why diseases have been identified for a mere 3% of genes in the human genome. Identification for de novo mutations as a cause of a disease requires a targeted molecular approach, which includes studying parents and affected subjects. The process for determining if the genetic basis of a disease may result in part from de novo mutations and the molecular approach to establish this link will be illustrated, using autism and schizophrenia as examples.

Watch this Scientific Journal Video about A Strategy to Identify de Novo Mutations in Common Disorders such as Autism and Schizophrenia at JoVE.com

A Strategy to Identify de Novo Mutations in Common Disorders such as Autism and Schizophrenia

Molecular genetic strategy for finding de novo mutations causing common disorders such as autism and schizophrenia.

There are several lines of evidence supporting the role of de novo mutations as a mechanism for common disorders, such as autism and schizophrenia. First, ...

a-strategy-to-identify-de-novo-mutations-common-disorders-such-as

Research

JoVE Journal

Medicine

Bilateral Common Carotid Artery Occlusion as an Adequate Preconditioning Stimulus to Induce Early Ischemic Tolerance to Focal Cerebral Ischemia

There is accumulating evidence, that ischemic preconditioning - a non-damaging ischemic challenge to the brain - confers a transient protection to a subsequent damaging ischemic insult. We have established bilateral common carotid artery occlusion as a preconditioning stimulus to induce early ischemic tolerance to transient focal cerebral ischemia in C57Bl6/J mice. In this video, we will demonstrate the methodology used for this study.

There is accumulating evidence, that ischemic preconditioning (PC) &#x2013; a non-damaging ischemic challenge to the brain - confers a transient protection to a subsequent damaging ischemic insult. We established bilateral common carotid artery occlusion (BCCAO) as a preconditioning stimulus to induce early ischemic tolerance (IT) to transient focal cerebral ischemia (induced by middle cerebral artery occlusion, MCAO) in C57Bl6/J mice.

There is accumulating evidence, that ischemic preconditioning - a non-damaging ischemic challenge to the brain - confers a transient protection to a ...

The In ovo CAM-assay as a Xenograft Model for Sarcoma

Sarcoma is a very rare disease that is heterogeneous in nature, all hampering the development of new therapies. Sarcoma patients are ideal candidates for personalized medicine after stratification, explaining the current interest in developing a reproducible and low-cost xenotransplant model for this disease. The chick chorioallantoic membrane is a natural immunodeficient host capable of sustaining grafted tissues and cells without species-specific restrictions. In addition, it is easily accessed, manipulated and imaged using optical and fluorescence stereomicroscopy. Histology further allows detailed analysis of heterotypic cellular interactions.
This protocol describes in detail the in ovo grafting of the chorioallantoic membrane with fresh sarcoma-derived tumor tissues, their single cell suspensions, and permanent and transient fluorescently labeled established sarcoma cell lines (Saos-2 and SW1353). The chick survival rates are up to 75%. The model is used to study graft- (viability, Ki67 proliferation index, necrosis, infiltration) and host (fibroblast infiltration, vascular ingrowth) behavior. For localized grafting of single cell suspensions, ECM gel provides significant advantages over inert containment materials. The Ki67 proliferation index is related to the distance of the cells from the surface of the CAM and the duration of application on the CAM, the latter determining a time frame for the addition of therapeutic products.

The In ovo CAM-assay as a Xenograft Model for Sarcoma

The in ovo chorioallantoic membrane (CAM) is grafted with fresh sarcoma-derived tumor tissues, their single cell suspensions, and permanent and transient fluorescently labeled established sarcoma cell lines. The model is used to study graft- (viability, Ki67 proliferation index, necrosis, infiltration) and host (fibroblast infiltration, vascular ingrowth) behavior.

Sarcoma is a very rare disease that is heterogeneous in nature, all hampering the development of new therapies. Sarcoma patients are ideal candidates for ...

Bladder Smooth Muscle Strip Contractility as a Method to Evaluate Lower Urinary Tract Pharmacology

We describe an in vitro method to measure bladder smooth muscle contractility, and its use for investigating physiological and pharmacological properties of the smooth muscle as well as changes induced by pathology. This method provides critical information for understanding bladder function while overcoming major methodological difficulties encountered in in vivo experiments, such as surgical and pharmacological manipulations that affect stability and survival of the preparations, the use of human tissue, and/or the use of expensive chemicals. It also provides a way to investigate the properties of each bladder component (i.e. smooth muscle, mucosa, nerves) in healthy and pathological conditions.
The urinary bladder is removed from an anesthetized animal, placed in Krebs solution and cut into strips. Strips are placed into a chamber filled with warm Krebs solution. One end is attached to an isometric tension transducer to measure contraction force, the other end is attached to a fixed rod. Tissue is stimulated by directly adding compounds to the bath or by electric field stimulation electrodes that activate nerves, similar to triggering bladder contractions in vivo. We demonstrate the use of this method to evaluate spontaneous smooth muscle contractility during development and after an experimental spinal cord injury, the nature of neurotransmission (transmitters and receptors involved), factors involved in modulation of smooth muscle activity, the role of individual bladder components, and species and organ differences in response to pharmacological agents. Additionally, it could be used for investigating intracellular pathways involved in contraction and/or relaxation of the smooth muscle, drug structure-activity relationships and evaluation of transmitter release.
The in vitro smooth muscle contractility method has been used extensively for over 50 years, and has provided data that significantly contributed to our understanding of bladder function as well as to pharmaceutical development of compounds currently used clinically for bladder management.

This manuscript presents a simple, yet powerful, in vitro method for evaluating smooth muscle contractility in response to pharmacological agents or nerve stimulation. Main applications are drug screening and understanding tissue physiology, pharmacology,&#160;and pathology.

We describe an in vitro method to measure bladder smooth muscle contractility, and its use for investigating physiological and pharmacological properties ...

Fundus Photography as a Convenient Tool to Study Microvascular Responses to Cardiovascular Disease Risk Factors in Epidemiological Studies

The microcirculation consists of blood vessels with diameters less than 150 &#181;m. It makes up a large part of the circulatory system and plays an important role in maintaining cardiovascular health. The retina is a tissue that lines the interior of the eye and it is the only tissue that allows for a non-invasive analysis of the microvasculature. Nowadays, high-quality fundus images can be acquired using digital cameras. Retinal images can be collected in 5 min or less, even without dilatation of the pupils. This unobtrusive and fast procedure for visualizing the microcirculation is attractive to apply in epidemiological studies and to monitor cardiovascular health from early age up to old age.
Systemic diseases that affect the circulation can result in progressive morphological changes in the retinal vasculature. For example, changes in the vessel calibers of retinal arteries and veins have been associated with hypertension, atherosclerosis, and increased risk of stroke and myocardial infarction. The vessel widths are derived using image analysis software and the width of the six largest arteries and veins are summarized in the Central Retinal Arteriolar Equivalent (CRAE) and the Central Retinal Venular Equivalent (CRVE). The latter features have been shown useful to study the impact of modifiable lifestyle and environmental cardiovascular disease risk factors.
The procedures to acquire fundus images and the analysis steps to obtain CRAE and CRVE are described. Coefficients of variation of repeated measures of CRAE and CRVE are less than 2% and within-rater reliability is very high. Using a panel study, the rapid response of the retinal vessel calibers to short-term changes in particulate air pollution, a known risk factor for cardiovascular mortality and morbidity, is reported. In conclusion, retinal imaging is proposed as a convenient and instrumental tool for epidemiological studies to study microvascular responses to cardiovascular disease risk factors.

Retinal image analysis is an unobtrusive procedure for visualizing the microcirculation. The impact of cardiovascular disease risk factors can result in changes of retinal vessel calibers. The procedures to acquire fundus images and the steps for calculating the vessel calibers are described.

The microcirculation consists of blood vessels with diameters less than 150 &#181;m. It makes up a large part of the circulatory system and plays an ...

Sublingual Immunotherapy as an Alternative to Induce Protection Against Acute Respiratory Infections

Sublingual route has been widely used to deliver small molecules into the bloodstream and to modulate the immune response at different sites. It has been shown to effectively induce humoral and cellular responses at systemic and mucosal sites, namely the lungs and urogenital tract. Sublingual vaccination can promote protection against infections at the lower and upper respiratory tract; it can also promote tolerance to allergens and ameliorate asthma symptoms. Modulation of lung&#8217;s immune response by sublingual immunotherapy (SLIT) is safer than direct administration of formulations by intranasal route because it does not require delivery of potentially harmful molecules directly into the airways. In contrast to intranasal delivery, side effects involving brain toxicity or facial paralysis are not promoted by SLIT. The immune mechanisms underlying SLIT remain elusive and its use for the treatment of acute lung infections has not yet been explored. Thus, development of appropriate animal models of SLIT is needed to further explore its potential advantages. 
This work shows how to perform sublingual administration of therapeutic agents in mice to evaluate their ability to protect against acute pneumococcal pneumonia. Technical aspects of mouse handling during sublingual inoculation, precise identification of sublingual mucosa, draining lymph nodes and isolation of tissues, bronchoalveolar lavage and lungs are illustrated. Protocols for single cell suspension preparation for FACS analysis are described in detail. Other downstream applications for the analysis of the immune response are discussed. Technical aspects of the preparation of Streptococcus pneumoniae inoculum and intranasal challenge of mice are also explained.
SLIT is a simple technique that allows screening of candidate molecules to modulate lungs&#8217; immune response. Parameters affecting the success of SLIT are related to molecular size, susceptibility to degradation and stability of highly concentrated formulations.

The present work illustrates the convenience of using sublingual immunotherapy to boost the innate immune response in the lungs and confer protection against acute pneumococcal pneumonia in mouse.

Sublingual route has been widely used to deliver small molecules into the bloodstream and to modulate the immune response at different sites. It has been ...

Using Adeno-associated Virus as a Tool to Study Retinal Barriers in Disease

M&#252;ller cells are the principal glial cells of the retina. Their end-feet form the limits of the retina at the outer and inner limiting membranes (ILM), and in conjunction with astrocytes, pericytes and endothelial cells they establish the blood-retinal barrier (BRB). BRB limits material transport between the bloodstream and the retina while the ILM acts as a basement membrane that defines histologically the border between the retina and the vitreous cavity. Labeling M&#252;ller cells is particularly relevant to study the physical state of the retinal barriers, as these cells are an integral part of the BRB and ILM. Both BRB and ILM are frequently altered in retinal disease and are responsible for disease symptoms.
There are several well-established methods to study the integrity of the BRB, such as the Evans blue assay or fluorescein angiography. However these methods do not provide information on the extent of BRB permeability to larger molecules, in nanometer range. Furthermore, they do not provide information on the state of other retinal barriers such as the ILM. To study BRB permeability alongside retinal ILM, we used an AAV based method that provides information on permeability of BRB to larger molecules while indicating the state of the ILM and extracellular matrix proteins in disease states. Two AAV variants are useful for such study: AAV5 and ShH10. AAV5&#160;has a natural tropism for photoreceptors but it cannot get across to the outer retina when administered into the vitreous when the ILM is intact (i.e., in wild-type retinas). ShH10 has a strong tropism towards glial cells and will selectively label M&#252;ller glia in both healthy and diseased retinas. ShH10 provides more efficient gene delivery in retinas where ILM is compromised. These viral tools coupled with immunohistochemistry and blood-DNA analysis shed light onto the state of retinal barriers in disease.

To investigate the blood-retinal barrier permeability and the inner limiting membrane integrity in animal models of retinal disease, we used several adeno-associated virus (AAV) variants as tools to label retinal neurons and glia. Virus mediated reporter gene expression is then used as an indicator of retinal barrier permeability.

M&#252;ller cells are the principal glial cells of the retina. Their end-feet form the limits of the retina at the outer and inner limiting membranes ...

Surgical Injury to the Mouse Pancreas through Ligation of the Pancreatic Duct as a Model for Endocrine and Exocrine Reprogramming and Proliferation

Expansion of pancreatic beta cells in vivo or ex vivo, or generation of beta cells by differentiation from an embryonic or adult stem cell, can provide new expandable sources of beta cells to alleviate the donor scarcity in human islet transplantation as therapy for diabetes. Although recent advances have been made towards this aim, mechanisms that regulate beta cell expansion and differentiation from a stem/progenitor cell remain to be characterized. Here, we describe a protocol for an injury model in the adult mouse pancreas that can function as a tool to study mechanisms of tissue remodeling and beta cell proliferation and differentiation. Partial duct ligation (PDL) is an experimentally induced injury of the rodent pancreas involving surgical ligation of the main pancreatic duct resulting in an obstruction of drainage of exocrine products out of the tail region of the pancreas. The inflicted damage induces acinar atrophy, immune cell infiltration and severe tissue remodeling. We have previously reported the activation of Neurogenin (Ngn) 3 expressing endogenous progenitor-like cells and an increase in beta cell proliferation after PDL. Therefore, PDL provides a basis to study signals involved in beta cell dynamics and the properties of an endocrine progenitor in adult pancreas. Since, it still remains largely unclear, which factors and pathways contribute to beta cell neogenesis and proliferation in PDL, a standardized protocol for PDL will allow for comparison across laboratories.

This protocol describes an injury model involving the surgical ligation of the pancreatic duct in the adult mouse pancreas, resulting in severe injury that establishes an environment that allows beta cell neogenesis and proliferation. This model can be used as a tool to study mechanisms involved in beta cell formation.

Expansion of pancreatic beta cells in vivo or ex vivo, or generation of beta cells by differentiation from an embryonic or adult stem cell, can provide ...

In Vivo Gene Transfer to the Rabbit Common Carotid Artery Endothelium

The goal of this method is to introduce a transgene into the endothelium of isolated segments of both rabbit common carotid arteries. The method achieves focal endothelial-selective transgenesis, thereby allowing an investigator to determine the biological roles of endothelial-expressed transgenes and to quantify the in vivo transcriptional activity of DNA sequences in large artery endothelial cells. The method uses surgical isolation of rabbit common carotid arteries and an arteriotomy to deliver a transgene-expressing viral vector into the arterial lumen. A short incubation period of the vector in the lumen, with subsequent aspiration of the lumen contents, is sufficient to achieve efficient and durable expression of the transgene in the endothelium, with no detectable transduction or expression outside of the isolated arterial segment. The method allows assessment of the biological activities of transgene products both in normal arteries and in models of human vascular disease, while avoiding systemic effects that could be caused either by targeting gene delivery to other sites (e.g. the liver) or by the alternative approach of delivering genetic constructs to the endothelium by germ line transgenesis. Application of the method is limited by the need for a skilled surgeon and anesthetist, a well-equipped operating room, the costs of purchasing and housing rabbits, and the need for expertise in gene-transfer vector construction and use. Results obtained with this method include: transgene-related alterations in arterial structure, cellularity, extracellular matrix, or vasomotor function; increases or reductions in arterial inflammation; alterations in vascular cell apoptosis; and progression, retardation, or regression of diseases such as intimal hyperplasia or atherosclerosis. The method also allows measurement of the ability of native and synthetic DNA regulatory sequences to alter transgene expression in endothelial cells, providing results that include: levels of transgene mRNA, levels of transgene protein, and levels of transgene enzymatic activity.

In Vivo Gene Transfer to the Rabbit Common Carotid Artery Endothelium

This method is to introduce a transgene into the endothelium of rabbit carotid arteries. Introduction of the transgene allows the assessment of the biological role of the transgene product either in normal arteries or disease models. The method is also useful for measuring activity of DNA regulatory sequences.

The goal of this method is to introduce a transgene into the endothelium of isolated segments of both rabbit common carotid arteries. The method achieves ...

An Experimental Model of Diet-Induced Metabolic Syndrome in Rabbit: Methodological Considerations, Development, and Assessment

In recent years, obesity and metabolic syndrome (MetS) have become a growing problem for public health and clinical practice, given their increased prevalence due to the rise of sedentary lifestyles and unhealthy eating habits. Thanks to animal models, basic research can investigate the mechanisms underlying pathological processes such as MetS. Here, we describe the methods used to develop an experimental rabbit model of diet-induced MetS and its assessment. After a period of acclimation, animals are fed a high-fat (10% hydrogenated coconut oil and 5% lard), high-sucrose (15% sucrose dissolved in water) diet for 28 weeks. During this period, several experimental procedures were performed to evaluate the different components of MetS: morphological and blood pressure measurements, glucose tolerance determination, and the analysis of several plasma markers. At the end of the experimental period, animals developed central obesity, mild hypertension, pre-diabetes, and dyslipidemia with low HDL, high LDL, and an increase of triglyceride (TG) levels, thus reproducing the main components of human MetS. This chronic model allows new perspectives for understanding the underlying mechanisms in the progression of the disease, the detection of preclinical and clinical markers that allow the identification of patients at risk, or even the testing of new therapeutic approaches for the treatment of this complex pathology.

We describe methods to develop an experimental model of diet-induced metabolic syndrome (MetS) in rabbits using a high-fat, high-sucrose diet. Animals developed central obesity, mild hypertension, pre-diabetes, and dyslipidemia, thus reproducing the main components of human MetS. This chronic model will allow acquisition of knowledge underlying mechanisms of disease progression.

In recent years, obesity and metabolic syndrome (MetS) have become a growing problem for public health and clinical practice, given their increased ...

A Rabbit Model of Durable Transgene Expression in Jugular Vein to Common Carotid Artery Interposition Grafts

Vein graft bypass surgery is a common treatment for occlusive arterial disease; however, long-term success is limited by graft failure due to thrombosis, intimal hyperplasia, and atherosclerosis. The goal of this article is to demonstrate a method for placing bilateral venous interposition grafts in a rabbit, then transducing the grafts with a gene transfer vector that achieves durable transgene expression. The method allows the investigation of the biological roles of genes and their protein products in normal vein graft homeostasis. It also allows the testing of transgenes for the activities that could prevent vein graft failure, e.g., whether the expression of a transgene prevents the neointimal growth, reduces the vascular inflammation, or reduces atherosclerosis in rabbits fed with a high-fat diet. During an initial survival surgery, the segments of right and left external jugular vein are excised and placed bilaterally as reversed end-to-side common carotid artery interposition grafts. During a second survival surgery, performed 28 days later, each of the grafts is isolated from the circulation with vascular clips and the lumens are filled (via an arteriotomy) with a solution containing a helper-dependent adenoviral (HDAd) vector. After a 20-min incubation, the vector solution is aspirated, the arteriotomy is repaired, and flow is restored. The veins are harvested at time points dictated by individual experimental protocols. The 28-day delay between the graft placement and the transduction is necessary to ensure the adaptation of the vein graft to the arterial circulation. This adaptation avoids rapid loss of transgene expression that occurs in vein grafts transduced before or immediately after grafting. The method is unique in its ability to achieve durable, stable transgene expression in grafted veins. Compared to other large animal vein graft models, rabbits have advantages of low cost and easy handling. Compared to rodent vein graft models, rabbits have larger and easier-to-manipulate blood vessels that provide abundant tissue for analysis.

This method describes the placement of interposition vein grafts in rabbits, the transduction of the grafts, and the achievement of durable transgene expression. This allows the investigation of physiological and pathological roles of transgenes and their protein products in grafted veins, and testing of gene therapies for vein graft disease.

Vein graft bypass surgery is a common treatment for occlusive arterial disease; however, long-term success is limited by graft failure due to thrombosis, ...