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Predisposition to hereditary renal carcinoma in the Eker rat involves a mutation of a putative tumor suppressor gene within chromosome band 10q12. We describe the identification of three unique polymorphic sequences in the vicinity of this locus following the microdissection, construction and characterization of a region-specific DNA library for rat chromosome band 10q12.
Fifty sequence-tagged sites (STSs) from human chromosome region, 11q13.4-->q25 were identified in microdissection-generated microclones. From a total of 124 single-copy microclones obtained from regions 11q14-->q22 and 11q23-->q25, 59 were sequenced, and a PCR primer pair was designed for each of them. Fifty of the 59 clones were mapped to regions ranging from 11q13.4 to 11q25 by means of PCR on a somatic hybrid cell panel with various deletions of the long arm of human chromosome 11. These STSs will contribute to the construction of physical cosmid/YAC maps representing the chromosome regions. They are also useful for analysis of chromosome aberrations, such as translocations, inversions or marker chromosomes.
We describe here the development of four sets of painting probes for mouse chromosomes 1 and 13, 2 and 8, 6 and 15, and X and Y by degenerate oligonucleotide-primed polymerase chain reaction (DOP-PCR) amplification of 10-20 copies of microdissected male meiotic chromosomes. The X,Y probe was obtained from the X-Y bivalent of diakinesis/metaphase I complements of mice with a normal karyotype, whereas the other probes were derived from tri- or quadrivalents in diakinesis/metaphase I of two reciprocal translocations, T(1;13)70H and T(2;8)2Wa, and one inversion heterozygote carrying a small deletion, In(6;15)Rb1Ald del6(15)1Wa. The specificity of these probes was established by fluorescence in situ hybridization (FISH) to meiotic and mitotic metaphase complements. The chromosomes painted by these probes could be identified by single or multicolor FISH.
To facilitate the identification of microsatellite genetic markers from a single swine chromosome, chromosome microisolation and microcloning have been used to generate a swine chromosome 6-specific DNA library. Ten copies of swine chromosome 6 were scraped from metaphase spreads, ligated to custom-prepared adaptors, and amplified by PCR. The purity of the amplified product was verified by fluorescent in situ hybridization. The utility of the chromosome painting probe for heterologous painting was demonstrated and confirmed that swine chromosome 6 is syntenic to human chromosomes 1p and 19q. A small insert genomic library of 1.39 x 10(6) clones was generated from the PCR-amplified chromosome 6 genomic DNA and screened for (GT)n microsatellite genetic markers. Nine (GT)n microsatellite markers were developed and genotyped on a Yorkshire x Meishan swine reference family. All nine markers genetically mapped to chromosome 6, confirming the purity of the microisolation method. The method used here should be adaptable to the microdissection of subchromosomal regions of not only the swine genome but also other livestock genomes.
K562 is a cell line with two acrocentric marker chromosomes containing abnormally banded regions (ABRs), derived from a Ph-positive chronic myelogenous leukemia (CML) patient. Using reverse and forward chromosome painting FISH analysis, we found that 9q34, 13q31, and 22q11 regions co-amplified in the ABRs-bearing acrocentric marker chromosomes of K562. Utilizing the ABRs of the cell line as target DNA for cDNA selection, we established a simple procedure for chromosome region-specific cDNA isolation. After first strand cDNA synthesis from fetal brain mRNAs, short fragment cDNAs (sf-cDNAs) were synthesized with a two-step amplification system by use of our modified Degenerate Oligonucleotide Primed Shuttle Polymerase Chain Reaction (DOP-Shuttle-PCR) method. The sf-cDNAs were hybridized onto RNase A treated metaphases from K562, and the ABRs were microdissected and reamplified with DOP-Shuttle-PCR primer-II. The reamplified sf-cDNAs were cloned into a pBluescript vector. Twenty randomly chosen clones were sequenced and classified into 8 groups. Three out of the 8 grouped clones had been mapped to the long arm of chromosome 22 (22q11), whereas the other 5 were novel cDNAs. Quantitative Southern blot analysis indicated that 7 out of the 8 grouped clones (87.5%) were derived from the co-amplified regions.
A strategy for rapid generation of chicken sex chromosome-Z painting probes has been developed using microdissection. Whole chromosome painting probes (WCPs) were prepared from 10-15 copies of mitotic metaphase chicken Z chromosomes. The microisolated chromosomes were subjected to PEG/proteinase K treatment in a collection drop to release DNA, which was then amplified using a degenerate oligonucleotide-primed shuttle PCR (DOP-Shuttle-PCR) strategy. Size distributions of the PCR products were analyzed by agarose gel electrophoresis and smears of DNA were revealed that ranged in size from 200-800 bp, without any evidence of preferential amplification. Both specificity and complexity of the probes have been analyzed by Southern blot and fluorescence in situ hybridization (FISH). Non-specific hybridization was efficiently blocked by using chicken competitor DNA. Analysis of the WCPs produced shows that collectively they provide uniform hybridization signals along the entire length of the chicken Z chromosome. To demonstrate one possible application of these complex probes, we screened a large-insert bacterial artificial chromosome (BAC) chicken genomic library to select Z chromosome-specific clones. To address specificity of the selected clones and to physically map them to the Z chromosome, FISH analysis was used. Of the 3 clones initially tested, one clone (C3) carrying a 250-kb insert mapped to the distal portion of the short arm of the chicken Z chromosome. Therefore, this technique has provided appropriate probes for screening large-insert genomic libraries. Further application of these probes includes the analysis of chromosome rearrangements, studies of cases of heteroploidy involving the Z chromosome, positional cloning of Z-linked genes and studies on mechanisms of sex-chromosome evolution in birds.
The neotropical rodents Akodon cursor (2n = 14, 15, and 16) and A. montensis (2n = 24 and 25), two closely related and morphologically indistinguishable species, have been compared by G-banding and chromosome painting. In situ hybridization of a biotinylated DOP-PCR product obtained from a microdissected chromosome of A. cursor onto A. montensis chromosomes was performed in combination with localization of telomeric sequences using a (TTAGGG)n oligomer as a FISH probe. The results provide evidence of the complex chromosomal rearrangements suggested by GTG-banding analysis, indicating the origin of one A. cursor autosome from three different A. montensis autosomes. Furthermore, the complete cytogenetic homeology between the A. cursor and A. montensis karyotypes was determined, along with the occurrence of tandem fusions and pericentric inversions and the loss of telomeres, centromeres, and chromosome arms. Evidence for the ancestral origin of the A. cursor karyotype is also provided.
The technique of microdissection and the structure of nephrons and collecting ducts from normal kidneys were studied. The structure of the assumedly normal nephrons and collecting ducts is characterized by the uniformity of the outlines of the tubules. The various parts of the tubules are distinguished by the differences in diameter and epithelial structure.
A unique opportunity presented itself for a morphologic study of experimental unilateral acute renal failure (ARF) in male rats. The ARF had been induced in the rats by temporary occlusion (1h) of the left renal artery. Twenty-nine rats were divided into subsets as follows: 2-3 h, 24 h, 1 week, 2, 4, 8, and 12 weeks following release of occlusion. Microdissection showed a heterogeneous population of abnormally structured proximal tubules in which the regressive lesions of tubular necrosis were combined with the progressive reaction of repair. The lesions demonstrated are reminiscent of those which have been described in ARF in the human and in experimental animals. Many proximal tubules in the 2- to 3-hour subset presented 1-3 disruptive lesions (DLs) while greater numbers of proximal tubules from the 24-hour group presented 1-5 DLs. Many proximal tubules presented no DLs, but nearly all from the 24-hour subset (97-100%) displayed a squamate appearance which paralleled and was caused by acute tubular necrosis. At 1 week, a dilated pars recta was common, but by this time, the squamate pattern had disappeared. Many casts were present. At 2 weeks, many fewer casts were present in proximal tubules and none were seen at 4, 8 or 12 weeks. The nephrons, particularly the proximal tubules, presented a variety of structural alterations at 2, 4, 8 and 12 weeks. Changes of special interest include (1) the presence of swan-necks; (2) a distinctive squamate appearance of the proximal tubules in the animals killed at 24 h; (3) a spiral, curled appearance caused by differential hyperplasia in animals at 4, 8 and 12 weeks, and (4) a tendency for ischemic lesions to involve all layers of the renal cortex.
Malformations of the cranial base, temporal bone and middle ear were induced in the offspring of Sprague-Dawley rats by a single intraperitoneal injection of 10-30 mg/kg etretinate (Tigasone) at days 8.5-10.5 of gestation. By differential staining of the embryonic craniofacial cartilage and bone, and microdissection of the otomandibular complex, the induced malformations were studied specifically. Defective formations of Meckel's cartilage and the cartilaginous skull base were found to be prominent features of the malformation. The malformation included defective middle-ear ossicles; especially the malleus and incus were fused with a shorter than normal long process and manubrium. In conjunction with the distal part of Meckel's cartilage, mandibular micrognathia was observed. All of the malformed tissues are derivatives of the first and second branchial arches. The teratogenically induced defects in the rat embryos show some similarities to the clinical syndromes of the first and second branchial arches in man.
The venous drainage of the testis was identified and described in an autopsy, radiographic and operative series of 30, 28 and 7 adult males, respectively. Testicular vein cast--right and left--was prepared in autopsy specimens to identify the course, tributaries and communications of the testicular vein. Intraoperative spermatic venography was done in 17 men with and in 11 men without varicocele. The retrograde spermatic venography showed the precise anatomy of the pampiniform plexus. Antegrade spermatic venography was done to identify the valves. During cystectomy in 7 adult men, the whole tree of the spermatic vein was taken and microdissected. Our findings show that the venous drainage of the testis cannot be looked upon as it is in the standard anatomy; on the contrary, it deviates from the description in the text. We demonstrated that the venous drainage of the testis is via the pampiniform plexus, which is primarily drained by the testicular and external pudendal veins. The testicular vein--midway between the internal inguinal ring and the lower pole of the kidney--divides into the medial and lateral branch to form a delta. The medial branch communicates with the ureteral and contralateral veins; there, it terminates in the left renal vein or inferior vena cava on the right side. The lateral branch communicates with colonic and renal capsular veins and terminates in the perinephric space. There is no cross-communication between the left and right testicular venous systems in the scrotal, retropubic or pelvic areas. The only cross-communication is in the abdomen, and is seen in only 50% of men. The testicular vein has no valves.
Circulating anucleate platelets contain minute amounts of residual megakaryocytic-derived mRNA. To study cell type-specific gene expression in platelets, an accurate and sensitive method to detect and quantify platelet mRNA that excludes contamination with leukocyte RNA is mandatory. Applying laser-assisted microdissection and manipulation (LMM) we could isolate platelets from hemalaun-stained cytospins under permanent visual control and after laser-photolysis of nucleated blood cells. For mRNA quantification, the platelet-specific mRNAs were subsequently measured by real time RT-PCR. High-copy beta3 integrin and low-copy a alpha2 integrin as well as tissue factor (TF) transcripts were analyzed in LMM-harvested platelets. In 91.2% (83/91) beta3 integrin was detectable with a mean threshold cycle (CT) value of 32.5+/-3.2 (< or =50,000 cells). The low-copy a 2 integrin mRNA was positive in 84.4% (38/45) with CT mean value of 36.9+/-1.3, indicating that the relative expression of alpha2 integrin mRNA in platelets was about 130 times lower than beta3. The TF transcript was undetectable in all samples. Comparing platelet mRNA from LMM isolation to that from limiting dilution series resulted in a high accordance for beta3 integrin transcript in both, recovery (91.2% vs. 95.2%) and CT value (32.5 vs. 32.8). These results demonstrate that the combination of LMM and real-time RT-PCR is a valuable tool for precise, platelet-specific mRNA analysis without contamination of other cells.
Adenosine plays several roles in the kidney mediated by the specific receptors A1, A2, and possibly A3. We studied the localization of adenosine A1 receptor mRNA in rat nephron segments using reverse transcription and polymerase chain reaction (RT-PCR). The nephron segments of male Sprague-Dawley rats (6 to 8 weeks old) were microdissected. Total RNA was prepared by the acid-guanidinium-phenol-chloroform method and used in the following RT-PCR assay. Because the PCR primers spanned no intron, samples reacted in the absence of RT were used as controls for amplification of genomic DNA. The PCR products were size-fractionated by electrophoresis, visualized with ethidium bromide staining, and confirmed by Southern blot analysis. PCR products were detected in all of the nephron segments examined. No signals were detected in samples reacted in the absence of RT. Strong signals were detected in glomeruli, medullary collecting duct, cortical thick ascending limb, and medullary thick ascending limb, while weak signals were found in proximal convoluted and straight tubules. Previously, the presence of A1 receptors has been demonstrated in glomeruli, collecting duct, and thick ascending limb in the rat kidney by autoradiography and binding studies. In addition to these segments, we further detected A1 receptor mRNA in proximal convoluted and straight tubules. Thus, A1 receptor mRNA seems to be broadly expressed along the nephron.
Nonciliated bronchiolar epithelial (Clara) cells, as both the primary target for metabolically activated pulmonary cytotoxicants and the progenitor during repair after bronchiolar injury, are critical for distal airway epithelial function and regeneration. The role of Clara cells in normal lung function is poorly understood partly because their abundance, sensitivity to cytotoxicants, and expression of differentiation markers vary by airway level and species. This study defines a strategy for maintenance in vitro of differentiated Clara cells within their local microenvironment. Lungs from adult mice were infalted with 1% agarose and distal airways were isolated by microdissection. Explants were cultured for 7 days in serum-free medium. Preservation of Clara cell morphology after 7 days in culture (DIC) was demonstrated using light and electron microscopy. Ciliated cells were also present. Cytochrome P450 monooxygenase activity, as measured by naphthalene epoxidation, was decreased 50% between 0 and 7 DIC, but the apparent stereoselectivity of metabolism was unchanged at 7 days. Marker proteins for differentiated Clara cells (secretory protein, CYP2F2 and CYP2B4) were detectable immunochemically throughout time in culture. Glutathione S-transferase activity and levels of reduced glutathione were unchanged over 7 DIC. We conclude that differentiated Clara cells can be maintained in cultures of explants from defined airway regions. Bronchiolar epithelial cells in this system are viable, synthesize and secrete secretory protein, metabolize xenobiotics via the cytochrome P450 system, have a stable phase II enzyme system, and maintain glutathione pools.
The surface epithelium lining the nasal airways is a potential target for inhaled contaminants such as ozone, endotoxin, formaldehyde, tobacco smoke, and organic dusts. The epithelial response to injury may depend on the toxicant, the type of epithelium, the severity of the injury, and the presence of inflammatory cells and their secreted products. To study mechanisms of toxicant-induced epithelial injury and repair, in the absence of cellular inflammation or other systemic effects, we have developed a culture system to maintain morphologically distinct nasal airway epithelium in vitro. Microdissected maxilloturbinates and proximal nasal septa of male F344/N rats were cultured at an air-liquid interface for up to 14 d in supplemented serum-free medium. Maxilloturbinates are lined by nonciliated cuboidal nasal transitional epithelium (NTE) with few or no mucous cells. The proximal nasal septum is lined by a mucociliary respiratory epithelium (RE) that normally contains numerous mucous cells. Preservation of the normal RE and NTE phenotype in culture was assessed by light and electron microscopy, and analysis of an airway mucin gene (rMuc-5AC) messenger RNA (mRNA). Both RE and NTE retained normal cell morphology for 14 d in culture (DIC). After 14 DIC there were 20% fewer RE cells in the septa (equal loss of ciliated and mucous cells) and 25% more NTE cells in the maxilloturbinates (increased number of basal cells). Compared with the RE, the NTE expressed consistently low levels of rMuc-5AC mRNA and had little to no histochemically detectable intraepithelial mucosubstances (IM) after 0, 3, 7, or 14 DIC. The amount of stored IM and the steady-state levels of rMuc-5AC mRNA in the RE decreased with time in culture. In summary, this culture system can maintain fully differentiated secretory and nonsecretory rat airway epithelia in vitro for up to 14 d. This study was an essential first step in developing a system to study the pathogenesis of toxicant-induced airway epithelial injury and mechanisms of cellular repair and adaptation in the absence of cellular inflammation and other systemic influences.
Terminal airways are affected in many lung diseases and toxic inhalations. To elucidate the changes in terminal airways in these diverse situations it will be helpful to profile and quantify gene expression in terminal bronchiolar epithelium. We used laser capture microdissection (LCM) to collect terminal bronchiolar epithelial cells from frozen sections of lungs of mice subjected to intratracheal bleomycin. The RNA from these cells was used for analysis of select messenger RNAs (mRNAs) by quantitative real-time polymerase chain reaction (PCR). In parallel, we used real-time PCR to analyze mRNAs in whole-lung homogenates prepared from other mice given intratracheal bleomycin. We found reductions of Clara cell-specific protein and keratinocyte growth factor receptor mRNAs in both terminal bronchiolar epithelium and whole-lung homogenates 7 d after bleomycin. In contrast, terminal bronchiolar epithelial transforming growth factor (TGF)-alpha mRNA was reduced but whole-lung TGF-alpha mRNA was not changed, whereas terminal bronchiolar epithelial epidermal growth factor (EGF) receptor mRNA was not changed but whole-lung EGF receptor was reduced. We conclude that LCM can isolate terminal bronchiolar epithelial cells for studies of cell-specific gene expression by quantitative real-time PCR, and that cell-specific gene expression in terminal bronchiolar epithelium is not necessarily reflected in analysis of whole-lung gene expression.
Recent studies have revealed that endothelins (ETs) have at least two types of receptors. One receptor has high affinity to ET-1 and ET-2 and low affinity to ET-3 (A type). The other receptor binds almost equally to ET-1, ET-2, and ET-3 (B type). In this study, microlocalization of mRNA coding for the A-type and B-type ET receptors was carried out in the rat kidney using a reverse transcription and polymerase chain reaction assay of individual microdissected renal tubule segments along the nephron, glomeruli, vasa recta bundle, and arcuate arteries. Large signals for the B-type receptor polymerase chain reaction product were detected in the initial and terminal inner medullary collecting duct and the glomerulus, while small signals were found in the cortical collecting duct and outer medullary collecting duct, vasa recta bundle, and arcuate artery. In contrast, A-type receptor mRNA was detected only in the glomerulus, vasa recta bundle, and arcuate artery. Thus, the two ET receptor subtypes are distributed differently along the nephron. This suggests that the two types of receptors and ET families may affect kidney functioning in different ways.
Stimulation of the release of nitric oxide (NO) in the kidney has been shown to result in renal hemodynamic changes and natriuresis. NO is a potent stimulator of soluble guanylate cyclase, leading to an increase of cyclic GMP. The precise localization of NO synthase and soluble guanylate cyclase in the renal structure is not known. In this study, the microlocalization of mRNAs coding for constitutive NO synthase and soluble guanylate cyclase was carried out in the rat kidney, using an assay of reverse transcription and polymerase chain reaction in individual microdissected renal tubule segments along the nephron, glomeruli, vasa recta bundle, and arcuate arteries. A large signal for constitutive NO synthase was detected in inner medullary collecting duct. Small signals were detected in inner medullary thin limb, cortical collecting duct, outer medullary collecting duct, glomerulus, vasa recta, and arcuate artery. Soluble guanylate cyclase mRNA is expressed largely in glomerulus, proximal convoluted tubule, proximal straight tubule, and cortical collecting duct, and in small amounts in medullary thick ascending limb, inner medullary thin limb, outer medullary collecting duct, inner medullary collecting duct, and the vascular system. Our data demonstrate that NO can be produced locally in the kidney, and that soluble guanylate cyclase is widely distributed in glomerulus, renal tubules, and the vascular system.