Protocol for chromosome-specific probe construction using PRINS, micromanipulation and DOP-PCR techniques

ABSTRACT Chromosome-specific probes have been widely used in molecular cytogenetics, being obtained with different methods. In this study, a reproducible protocol for construction of chromosome-specific probes is proposed which associates in situ amplification (PRINS), micromanipulation and degenerate oligonucleotide-primed PCR (DOP-PCR). Human lymphocyte cultures were used to obtain metaphases from male and female individuals. The chromosomes were amplified via PRINS, and subcentromeric fragments of the X chromosome were microdissected using microneedles coupled to a phase contrast microscope. The fragments were amplified by DOP-PCR and labeled with tetramethyl-rhodamine-5-dUTP. The probes were used in fluorescent in situ hybridization (FISH) procedure to highlight these specific regions in the metaphases. The results show one fluorescent red spot in male and two in female X chromosomes and interphase nuclei.

Use of Three-Dimensional Curved-Multiplanar Reconstruction Images for Sylvian Dissection in Microsurgery of Middle Cerebral Artery Aneurysms

PURPOSE: The purpose of this study was to introduce a method of using three-dimensional (3D) curved-multiplanar reconstruction (MPR) images for sylvian dissection during microsurgical treatment of middle cerebral artery (MCA) aneurysms. MATERIALS AND METHODS: Forty-nine patients who had undergone surgery for MCA aneurysms were enrolled. We obtained the 3D curved-MPR images along the sphenoid ridge using OsiriX MD™ imaging software, compared sylvian dissection time according to several 3D MPR image factors, and investigated the correlations between these images and intraoperative findings. RESULTS: Utilizing preoperative information of the sylvian fissure (SF) and peri-aneurysmal space on 3D curved-MPR images, we could predict the feasibility of sylvian dissection for a safe surgery. 3D curved-MPR images showed several features: first, perpendicular images to the sylvian surface in the same orientation as the surgeon's view; second, simultaneous visualization of the brain cortex, vessels, and cisternal space; and third, more accurate measurement of various parameters, such as depth of the MCA from the sylvian surface and the location and width of the SFs. CONCLUSION: In addition to conventional image studies, 3D curved-MPR images seem to provide useful information for Sylvian dissection in the microsurgical treatment of MCA aneurysms.

Phacoemulsification in posterior polar cataract / Facoemulsificação na catarata polar posterior

ABSTRACT Purpose: To evaluate the results and complications of phacoemulsification surgery in eyes with posterior polar cataracts and compare the techniques of viscodissection and hydrodissection. Methods: The medical records of 29 consecutive patients (16 males, 13 females) with posterior polar cataracts (44 eyes) who had undergone cataract surgery were retrospectively reviewed. Patients were divided into two groups according to the technique used; viscodissection was applied to the experimental group (group 1) and hydrodissection to the control group (group 2). Results: The postoperative best corrected visual acuity (BCVA) was 0.19 ± 0.22 logMAR (mean ± standard deviation) (range 0.00-0.70) in group 1 and 0.25 ± 0.18 logMAR (range 0.00-0.70) in group 2. Although the mean postoperative BCVA in group 1 was greater than that in group 2, the difference was not statistically significant (p=0.165). The mean postoperative BCVA was significantly greater than the mean preoperative BCVA in both groups (p=0.00). Intraoperatively, posterior capsular rupture occurred during the removal of the cortex in three eyes (13%) of group 1 patients, with vitreous loss and anterior vitrectomy in one eye only. In group 2, six eyes (28.5%) presented posterior capsular rupture, and anterior vitrectomy was performed because of vitreous loss in three eyes. Although the percentage of posterior capsular rupture was greater in group 2, the difference was not statistically significant (p=0.207). Conclusions: Complications in posterior polar cataract surgeries can be overcome by being careful throughout the surgery and using proper techniques. Viscodissection may be better for avoiding posterior capsular rupture than hydrodissection.

RESUMO Objetivo: Avaliar os resultados e complicações da cirurgia de facoemulsificação em olhos com catarata polar posterior e comparar as técnicas de viscodissecção e hidrodissecção. Métodos: Os prontuários de 29 pacientes consecutivos (16 homens, 13 mulheres) com posterior cataratas polares (44 olhos), que haviam sido submetidos a cirurgia de catarata foram analisados retrospectivamente. Os pacientes foram divididos em dois grupos de acordo com a técnica utilizada; viscodissecção foi aplicada ao grupo experimental (grupo 1) e hidrodissecção para o grupo de controle (grupo 2). Resultados: No pós-operatório, a melhor acuidade visual corrigida (BCVA) foi 0,19 ± 0,22 logMAR (média ± desvio padrão) (variação 0,00-0,70) no grupo 1 e 0,25 ± 0,18 (0,00-0,70) logMAR no grupo 2. Embora a média da BCVA pós-operatória do grupo 1 tenha sido maior do que a do grupo 2, a diferença não foi estatisticamente significativa (p=0,165). A melhor acuidade visual corrigida pós-operatória foi significativamente melhor do que no pré-operatório, em ambos os grupos (p=0,00). No grupo 1, houve ruptura capsular posterior durante a remoção do córtex em três olhos (13%); houve perda vítrea e necessidade de vitrectomia anterior mas apenas em destes olhos. No grupo 2, a ruptura da cápsula posterior ocorreu em seis olhos (28,5%); vitrectomia anterior foi necessária após a perda vítrea em três destes olhos. Embora a porcentagem de ruptura da cápsula posterior tenha sido maior no grupo 2, a diferença não foi estatisticamente significativa (p=0,207). Conclusões: As complicações em cirurgias de catarata polar posterior podem ser superadas com cautela durante toda a cirurgia e usando técnicas adequadas. Viscodissecção é melhor para evitar a posterior ruptura capsular do que hidrodissecção.

Complete removal of the spinal nerve sheath tumors. Surgical technics and results from a series of 30 patients / Remoção completa dos tumores da bainha dos nervos raquianos. Técnica cirúrgica e resultados de uma série de 30 pacientes

Objective: Observe whether a microsurgical gross total removal (GTR) of a spinal nerve sheath tumors (SNSTs) is safe and decreases the tumor recurrence. Method: We identify 30 patients with 44 SNSTs. Results: We operated upon 15 males and 15 females patients; mean age 40 years. GTR was achieved in 29 (96.6%) instances. Surgical mortality was 3.3% and the recurrence rate was 3.3%. The median follow-up time was 6.2 years. Conclusion: The surgical approach used in this group of patients afford that the great majority of tumors could be totally removed with low mortality and low recurrence rates, proving to be safe and effective. .

Objetivo: Observar se a ressecção microcirúrgica completa dos shwannomas ou neurofibromas raquianos é uma técnica segura e efetiva. Método: Foram operados 30 pacientes com 44 schwannomas ou neurofibromas intrarraquiano. Resultados: A remoção total da lesão ocorreu em 27 casos (96.6%). A taxa de mortalidade cirúrgica observada nesta série foi de 3.3%. O tempo médio de seguimento foi de 6.2 anos. Conclusão: A estratégia microcirúrgica empregada com esses pacientes propiciou a remoção total dos tumores na maioria dos pacientes, com baixa mortalidade e recidiva tumoral, mostrando ser segura e efetiva. .

Anatomical study of the pigs temporal bone by microdissection

PURPOSE: Initial study of the pig`s temporal bone anatomy in order to enable a new experimental model in ear surgery. METHODS: Dissection of five temporal bones of Sus scrofa pigs obtained from UNIFESP - Surgical Skills Laboratory, removed with hole saw to avoid any injury and stored in formaldehyde 10% for better conservation. The microdissection in all five temporal bone had the following steps: inspection of the outer part, external canal and tympanic membrane microscopy, mastoidectomy, removal of external ear canal and tympanic membrane, inspection of ossicular chain and middle ear. RESULTS: Anatomically it is located at the same position than in humans. Some landmarks usually found in humans are missing. The tympanic membrane of the pig showed to be very similar to the human, separating the external and the middle ear. The middle ear`s appearance is very similar than in humans. The ossicular chain is almost exactly the same, as well as the facial nerve, showing the same relationship with the lateral semicircular canal. CONCLUSION: The temporal bone of the pigs can be used as an alternative for training in ear surgery, especially due the facility to find it and its similarity with temporal bone of the humans. .

Microdissection Testicular Sperm Extraction (micro-TESE) as a Sperm Acquisition Method for Men with Nonobstructive Azoospermia Seeking Fertility: Operative and Laboratory Aspects

Introduction: Rare foci of sperm production may be found in up to 60% of men with nonobstructive azoospermia (NOA). Sperm production, if present, is minimal for sperm appearance in the ejaculate. Given that there are no treatment options to restore fertility, sperm retrieval is the only alternative to find testicular sperm than then can be used for in vitro fertilization (IVF). Among sperm acquisition methods, micro-TESE has higher success rates at obtaining sperm compared with testicular sperm extraction and testicular sperm aspiration. Materials and Methods: This video describes the operative aspects of micro-TESE, performed on an outpatient basis, in a man with NOA and history of cryptorchidism in whom orchidopexy was performed at age 6. The concept of micro-TESE is to identify areas of sperm production within the testes with the aid of optical magnification (15-25X) and based on the size and appearance of the seminiferous tubules (ST). Conclusion: Micro-TESE allowed the identification and extraction of sperm-containing STs with minimum tissue excision and marked reduction in time processing of testicular specimens for sperm injection.

Microsurgical ressection for parasagittal meningiomas with preservation of the parasagittal sinus and excelent neurovascular control / Ressecção microcirúrgica dos meningiomas parasagitais com excelente controle vascular e preservação do seio longitudinal superior

Objective: It was to observe whether a microsurgical gross total removal (GTR) of a parasagittal meningioma (PSM) outside the sinus is a safe and efficient procedure. Method: We identify 58 parasagittal meningiomas patients. Medical charts, operative reports, imaging studies and clinical follow-up evaluations were reviewed. Results: GTR of the mass was achieved in 45 (77.7%) instances. The surgical mortality rate was 1.7%. The median follow-up time was 63 months. Conclusion: The surgical approach used in this group of patients afford that the great majority of tumors could be totally removed with low mortality, proving to be safe and effective. .

Objetivo: Foi observar se a ressecção microcirúrgica extrassinusal dos meningiomas parasagitais é uma técnica segura e efetiva. Método: Foram estudados 58 pacientes portadores de meningiomas parasagitais. Quadros médicos, relatórios de operações, exames de imagem e seguimento clínico foram revisados. Resultados: A remoção total da lesão, fora do seio, ocorreu em 45 casos (77,7%). A taxa de mortalidade cirúrgica observada nesta série foi de 1,7%. O tempo médio de seguimento foi de 63 meses. Conclusão: A estratégia microcirúrgica empregada propiciou a remoção total dos meningiomas na maioria dos pacientes, com baixa mortalidade e morbidade, mostrando ser segura e efetiva. .

Molecular approach of the fragile chromosomal region Xq31-34 in cattle (Bos taurus) by microdissection and DOP-PCR / Aproximação molecular da região cromossômica frágil Xq31-34 em bovinos (Bos taurus) utilizando microdissecação cromossômica e DOP-PCR

Fragile sites (FS) are chromosomal regions where the normal compactation of chromatine is not observed. FRAXA (Fra Xq27.3, X sexual chromosome) is one of the most studied FS in humans. FRAXA is an expansion of the trinucleotide CGG located in the gene FMR-1. In cattle, sites of chromosomal fragility were reported in BTAX, associated with different pathologies and fertility impairment. Chromosomal microdissection has became a valuable tool for isolating chromatine fragments. In this work, it was combined the chromosomal microdissection technique with DOP-PCR in order to carry out a molecular analysis of the fragile chromosomal region BTAXq31-34. In that region, polymorphic DNA-RAPD sequences (GC rich) are present and sequences of the gene FMR-1 are missing. The results showed the usefulness of the microdissection-DOP-PCR technique for molecular characterization of fragile chromosomal sites in cattle.

Os sítios frágeis (FS) são regiões de cromossomo onde a compactação normal da cromatina não é realizada. O FRAXA (Fra Xq27.3, cromossomo sexual X) é um dos FS mais estudados em seres humanos. O FRAXA apresenta expansão do trinucleotídeo CGG localizado no gene FMR-1. Em bovinos, existem estudos informando sobre fragilidade cromossômica em BTAX associada com diversas patologias e alterações na fertilidade. A microdissecação cromossômica é uma valiosa técnica para isolar fragmentos de cromatina. Neste trabalho, combinou-se a técnica de microdissecação de cromossomo com DOP-PCR para executar a análise molecular da região do sitio frágil cromossômico BTAXq31-34. Naquela região estão presentes seqüências do polimorfo DNA-RAPD (rico em GC), em que as seqüências do gene FMR-1 estão ausentes. Os resultados mostram a utilidade da técnica de microdissecação-DOP-PCR para a caracterização molecular de sítios frágeis cromossômicos em bovinos.

Análise do perfil de expressão gênica de carcinoma ductal in situ e invasivo em tumores de mama através da técnica de microarray / Gene expression profile analysis of ductal carcinoma in situ and invasive of the breast cancer by Microarray techonology

O câncer de mama está entre as neoplasias de maior incidência e é responsável pela alta taxa de mortalidade entre as mulheres no mundo todo. O carcinoma ductal é o tipo histológico mais freqüente. O carcinoma ductalin situ (DCIS) inclui um grupo de tumores de mama pré-invasivos com potencial maligno distinto, podendo progredir rapidamente para carcinoma invasivo ou não apresentar evolução durante um longo período da doença. ... Neste estudo foram analisados 40 casos de mama, sendo 5 amostras de tecido de mamário não neoplásico(N), 16 casos de amostras pareadas de carcinoma ductal (in situ e invasivo), 5 DCIS puro, 9 DCIS coexistindo com o componente invasor (DCIS/IDC) e 5 carcinomas ductais invasivos (IDC). .... Dois delineamentos experimentais foram usados: comparação entre lesões in situ e invasivo da mesma amostra (DCIS e IDC); e comparação de grupos de lesões que mimetizam a progressão de câncer ductal de mama, utilizando amostras independentes [células epiteliais de mama capturadas de amostra não neoplásica (N), células tumorais capturadas das lesões: DCIS puro, DCIS/IDC e IDC]. ... Na comparação entre as 3 lesões (DCIS puro, DCIS/IDC e IDC), o DCIS puro mostrou maior divergência molecular, contradizendo os aspectos morfológicos. ... Esse resultado confirmou que o padrão de expressão entre essas lesões é semelhante mesmo avaliando genes sabidamente envolvidos no processo. ... Assim, baseado nos dados desse estudo, demonstramos que as células do carcinoma ductal in situ que coexistem com as células do carcinoma invasivo apresentam alterações moleculares antes da modificação morfológica da lesão, e isso pode ser explorado para se identificar genes alterados que possam predizer a capacidade de invasão. Além disso, esse estudo identificou vários genes candidatos a marcadores moleculares de prognóstico e também preditivos do risco de progressão de doença não invasiva.

Breast carcinoma is one of the most incidence neoplasias and is responsible for a high death-rate among women worldwide. The ductal carcinoma is the most frequent histological type. Ductal carcinoma in situ (DCIS) includes a group of preinvasive breast tumors with distinct malignant potentials. DCIS can have different outcomes, progressing rapidly to invasive cancer or slowly changing over a long period of the disease. Lately, one of the most challenges in molecular research in this field is to identify genes able to predict the risk of progression to invasive disease and prognostic marker. In this study were used 40 breast cases, being 5 non-neoplasic mammary tissues samples (N), 16 matched pairs of ductal carcinoma (in situ and invasive), 5 pure DCIS, 9 DCIS coexisting with invasive ductal carcinoma (DCIS/IDC) e 5 IDC. The RNA from epithelium cells laser capture microdissected were amplified and hybridized using reference design with dye swap in two distinct customized cDNA microarrays platforms. One containing 4.608 cDNA that represent human genes (4.8K) and other containing 390 genes belonging to WNT, PI3K signaling pathways and EMT process (Epithelial-mesenchymal transition). Two designing assays were used: comparisons between lesions in situ and invasive from the same patient (DCIS e IDC) and comparison among lesion groups that mimics the progression of breast ductal carcinoma using independent samples (breast epithelium cells microdissected of non-neoplasic tissues (N), tumoral cells microdissected of lesions: DCIS puro, DCIS/IDC e IDC. In the 4.8k platform, 16 matched-pair samples were used to compare the tumor cells expression profile of DCIS and IDC from the same patient. It was identified 33 candidates differentially expressed (t de Student pairwise - Fold >⎜1,5⎥ e pvalue<0,01), being candidates' genes to be involved in transition from DCIS to IDC. To 4 (LUM, RDH-E2, CXCL13 e POSTN) of 8 selected genes was confirmed differential expression through quantitative RT-PCR (qRT-PCR). Two genes (LUM and CRABP2) over expressed in IDC were selected to verify association with others molecular markers and/or clinicopathological parameters through Tissue Microarray. The LUM protein expression showed positive association with CKs 5/6, CK 14, CK8 e 18 and HER2/neu positive, while CRABP2 showed positive association with ER, PR, CK8, CK18, luminal A, p53 and negatively with CK14. Seeking to characterize molecular aspects of ductal carcinoma in situ of the breast progression, the general gene expression profile among 4 groups that mimics the progression was analyzed (N, DCIS pure, CIS/IDC e IDC) performing ANOVA test (pFDR<0,01) and followed by Tukey´s test, being characterized as most diverge the N group, as expected. The comparison among the 3 lesions (DCIS pure, DCIS/IDC e IDC), the pure DCIS showed the most molecular divergence, contradicting the morphological aspects. To identify genes able to predict the potential risk of invasion of DCIS, it was compared the expression profile of two morphologically identical lesions (DCIS pure e DCIS/IDC), identifying 147 genes (ANOVA - Fold >⎜2⎥ e pFDR<0,01). Hierarchical cluster based on expression profile of those genes could segregate the samples into two distinct groups in which 100% of non-neoplasic samples and 60% of pure DCIS remained in the same group and discriminated from DCIS/IDC (100%). Five (C16orf5, SULF-1, LOX, GOSR2 and TXNL2) candidates were confirmed through qRT-PCR as predict markers of DCIS progression. To assess the functional aspects of invasion process, it was used a platform containing genes belonging to WNT, PI3K signaling pathways and EMT process. The same experimental designing was used for 10 matched-pair, resulting in 32 differentially expressed genes between DCIS e IDC (t de Student pairwise - pvalue <0,05). This result confirmed that expression profile between the lesions is similar even assessing genes involved in this process. Between pure DCIS and DCIS/IDC were identified 15 differentially expressed genes (Wilcoxon ­ p<0,05) whose expression profile could segregate the samples in the same way as for the 147 genes. Triple classifiers were built seeking to segregate the DCIS pure and DCIS/IDC samples. One gene triple belonged to each signaling pathways, WNT (CSNK1A1L, LRP3 and SDC2), PI3K (PLCG2, INPP1 and DGKA) and EMT process (HDGF, CDH13 e TWIST1) were able to segregate the samples being predictor candidates of DCIS progression. Hence, based on this study data, we showed that ductal carcinoma in situ coexisting with invasive ductal carcinoma presents molecular alteration before lesion morphological modifications, and this may be exploited to identify altered genes able to predict invasion capacity. Furthermore, this study identified many candidate genes to molecular markers for outcome and also predicting the risk of noninvasive disease progression.

Microdissecção e captura a laser na investigação do gene TP53 em tecidos incluídos em parafina / Laser-capture microdissection for TP53 gene analysis in paraffin-embedded tissues

INTRODUÇÃO: Microdissecção e captura a laser (MCL) é uma técnica de desenvolvimento recente que permite a coleta de células individuais ou pequeno conjunto de células para análise molecular. Atualmente, no Brasil, há raros microscópios para MCL, de modo que a divulgação dos procedimentos inerentes a essa técnica é oportuna para destacar seu amplo potencial para diagnóstico e investigação. OBJETIVO: Este trabalho descreve a padronização dos procedimentos de MCL e de extração de DNA de material fixado em formalina e incluído em parafina. MATERIAL E MÉTODOS: Foram estudados o éxon 8 do gene TP53 e o gene da ciclofilina em amostras de tecido normal e de neoplasias de fígado e rim provenientes de modelo de carcinogênese química induzida em rato. A extração do DNA foi comprovada por reação em cadeia da polimerase (nested-PCR). RESULTADOS: Foram padronizados os procedimentos de preparo dos cortes histológicos, de microdissecção e captura a laser e de obtenção de seqüências gênicas pela reação de nested-PCR para tecidos incluídos em parafina. Obtivemos amplificação de 48,3 por cento das amostras para o éxon 8 do gene TP53 e 51,7 por cento para o gene da ciclofilina. Considerando pelo menos um dos dois segmentos gênicos, foram amplificadas 79,3 por cento das amostras. DISCUSSÃO E CONCLUSÃO: A extração de DNA de tecidos fixados em formalina e incluídos em parafina e a técnica de nested-PCR foram adequadamente padronizadas para produtos gênicos de interesse, obtidos de material coletado por MCL. Esses procedimentos podem ser úteis para a obtenção de seqüências de DNA de arquivos para análise molecular.

BACKGORUND: Laser-capture micro-dissection (LCM) is a recently developed procedure that provides single cells or specific cell groups for molecular analysis. Currently, there are few LCM systems in Brazil, in such a way that it is necessary to disseminate the technical procedures inherent to the methodology, and also to characterize its enormous potential for diagnosis and research. OBJECTIVE: This study describes the standardization of LCM and DNA extraction from formalin fixed and paraffin-embedded tissues. MATERIAL AND METHOD: The gene TP53 exon 8 and the cyclophilin gene were studied in normal and neoplastic liver and kidney samples from a chemical carcinogenesis model in rat. DNA extraction was confirmed by nested-PCR. RESULTS: Histological sections preparation for LCM and the nested-PCR procedures were standardized; 48.3 percent amplifications of the gene TP53 exon 8 and 51.7 percent of the cyclophilin gene samples were obtained. When at least one of the gene segments was considered, 79.3 percent samples presented amplification. DISCUSSION AND CONCLUSION: Procedures for DNA extraction from formalin fixed and paraffin-embedded tissues collected by LCM were standardized. They can be useful for DNA collection for molecular studies.

DNA genotyping of oral epithelial cells by laser capture microdissection / 法医学杂志

OBJECTIVE@#The STR genotypping of trace oral epithelial cells which are microdissected by laser capture microdissection system (LCM) is explored.@*METHODS@#The oral epithelial cells are microdissected using a low-power infrared laser by VERITAS Microdissection Instrument. STR loci of Profiler Plus are detected by multiplex PCR procesures.@*RESULTS@#DNA genotyping of 7-8 oral epithelial cells are succeeded, and DNA genotyping of 3-4 oral epithelial cells are failed.@*CONCLUSION@#It is viable in genotyping of trace oral epithelial cells by Laser Capture Microdissection as a new technology of seperating single cell.

Microsurgical anatomy of the arterial compartment of the cavernous sinus: analysis of 24 cavernous sinus

O seio cavernoso é estrutura complexa localizada de cada lado da sela túrcica, sendo seu conhecimento microanatômico indispensável quando se considera abordar cirurgicamente esta região. Estudaramos em laboratório de microcirurgia a microanatomia dos componentes arteriais deste espaço em 24 seios cavernosos, sendo que em todos a artéria carótida interna estava preenchida com látex colorido. O tronco meningo-hipofisário esteve presente em 18 casos (75%). Quando ausente, as artérias constituintes deste tronco se originaram diretamente das artérias carótidas internas (ACIs) intracavernosas. Quando presente, em 14 casos (77,7%), estavam trifurcados e em 4 casos (23,3%) bifurcados. A artéria tentorial foi identificada em todos os casos, porém sua origem foi variada, ocorrendo no tronco meningo-hipofisário em 17 casos (70,8%) e na artéria carótida interna intracavernosa em 7 casos (29,1%). Em 1 caso verificou-se a presença de uma artéria tentorial acessória. A artéria meningéia dorsal estava presente em 22 casos (91,6%) e ausente em 2 casos (8,4%). Nos seios cavernosos onde a mesma foi identificada, a sua origem ocorreu no tronco meningo-hipofisário em 17 casos (77,2%), da ACI intracavernosa em 4 casos (18,1%) e da artéria hipofisária inferior em 1 caso (4,1%). A artéria hipofisária inferior foi identificada em todos os casos, tendo sua origem no tronco meningo-hipofisário em 16 (66,6%) e na ACI intracavernosa em 8 (33,3%) casos. A artéria inferior do seio cavernoso ou tronco ínfero-lateral foi isolada em 100% dos casos e em todos se originou da ACI intracavernosa. A artéria de McConnell não foi identificada em nenhum seio cavernoso.

Heterochromatin and chromosome evolution: a FISH probe of Cebus apella paraguayanus (Primate: Platyrrhini) developed by chromosome microdissection

Neotropical Primate karyotypes are highly variable, particularly in the heterochromatic regions, not only regarding the amount of heterochromatin, but also the composition. G and C banding and FISH techniques provide useful information to characterize interspecific relationships. We used chromosome microdissection to develop a FISH probe of the chromosome 11 heterochromatic block (11qHe+) of Cebus apella paraguayanus (CAPp). Fragments of the 11qHe+ microdissected from fibroblast cell culture were collected in a PCR tube, amplified by degenerate oligonucleotide primer-PCR and subsequently labeled. The specificity of the FISH probe was confirmed in metaphases of some Ceboidea species. Signals were located in the He+ of chromosomes 4, 11, 12, 13, and 19 of CAPp and in the He+ of chromosomes 4, 12 and 13 of C. a. nigritus (CAPn); no signals were observed when other Ceboidea species were analyzed. We propose that the heterochromatin observed in CAPp and CAPn is specific for these species. We consider this C. apella heterochromatin identity as a possible key for the interpretation of chromosomal evolution in these Ceboidea.