Primed In Situ Research Articles

CAPS DNA Marker Labeling Using Primed in Situ Hybridization Technique for Chromosome of Strawberry (Fragaria × ananassa Duch.)

ABSTRACTThe cleavage amplified polymorphic sequence (CAPS) markers were developed to identify strawberry cultivars from unknown samples. These markers seemed to have a chromosome specificity. However, no reports confirmed it. The present study was conducted to verify the location of a CAPS marker on the chromosomes using a primed in situ (PRINS) technique. A CAPS marker was hybridized with the ‘Sachinoka’ chromosomes using the PRINS technique. The samples were observed under a fluorescence microscope. When the chromosomes were hybridized with the single marker, fluorescent signals were found on two chromosomes. From the results, the CAPS marker was confirmed to have a chromosome specificity by chromosome observation.

Mapping of rRNA genes and telomeric sequences in Danube salmon (Hucho hucho) chromosomes using primed in situ labeling technique (PRINS)

In the current paper we described the application of primed in situ (PRINS) labeling approach for the chromosomal mapping of repetitive DNA sequences in Danube salmon (Hucho hucho) (2n = 82, NF = 112). PRINS was successfully performed with primers enabling amplification of 5S rRNA genes (minor rDNAs), NOR building DNA sequences (major rDNAs), and telomeric sequences. Two loci of 5S rRNA were observed on distinct chromosome pairs; the minor arrays were located interstitially on the long (q) arms of two large metacentrics (chromosomes No. 3) and the large clusters of 5S rDNAs were assigned to the short (p) arms of two subtelocentric chromosomes No. 18. Major rDNA clusters were observed on the p-arms of two submeta-subtelocentric chromosomes No. 10. These chromosomal areas were built with GC-rich chromatin what was proved in the course of chromomycin A(3) (CMA(3)) staining performed sequentially. Major and minor rDNA families were not co-localized in the Danube salmon chromosomes. The distinct hybridization signals at the ends of all the chromosomes were provided in the course of PRINS with (CCCTAA)(n) primer. The chromosomal localization of rRNA genes and telomeric DNA sequences was discussed in the context of Salmonidae karyotype evolution.

Multicolor PRINS and multicolor PNA

Both PRimed IN Situ (PRINS) and Peptide Nucleic Acid (PNA) technologies have emerged as research techniques, but they have quickly evolved to applications in biological diagnosis assays. The two procedures now constitute efficient alternatives to the conventional fluorescence in situ hybridization (FISH) procedure for in situ chromosome identification and aneuploidy detection. They present several advantages (specificity, speed, discriminating ability) that make them very attractive for a number of cytogenetic purposes. Multicolor PRINS and PNA protocols have been described for the specific identification of human chromosomes. Various applications have already been developed in human genetics and new adaptations are ongoing.

In situ aneuploidy assessment in human sperm: the use of primed in situ and peptide nucleic acid-fluorescence in situ hybridization techniques

Both the primed in situ (PRINS) and the peptide nucleic acid-fluorescence in situ hybridization (PNA-FISH) techniques constitute alternatives to the conventional (fluorescence in situ hybridization, FISH) procedure for chromosomal investigations. The PRINS reaction is based on the use of a DNA polymerase and labeled nucleotide in an in situ primer extension reaction. Peptide nucleic acid probes are synthetic DNA analogs with uncharged polyamide backbones. The two procedures present several advantages (specificity, rapidity and discriminating ability) that make them very attractive for cytogenetic purposes. Their adaptation to human spermatozoa has allowed the development of new and fast procedures for the chromosomal screening of male gametes and has provided efficient complements to FISH for in situ assessment of aneuploidy in male gametes.

From the conception of the PRINS to its coronation

As a non-isotopic molecular cytogenetic technique, the primed in situ (PRINS) labelling reaction represents a major technological progress achieved in the past decade. It has become a routine technique for the microscopic visualization of specific DNA sequences in cells and nuclei and constitutes a good alternative to the fluorescence in situ hybridization (FISH) procedure. Among the multiple advantages that characterize the PRINS technique, specificity, rapidity, reliability, reproducibility, and cost-effectiveness can be mentioned. PRINS can be in addition associated with other techniques like FISH, indirect immunofluorescence, and nick translation. The most recent developments show the great potential of this technique. Now PRINS can be used to study single-copy genes and, consequently, can be routinely used to investigate deletions associated with microdeletion syndromes. Therefore, the PRINS technique has the potential to become a widely used molecular cytogenetic tool in clinics and research. This short review presents how the PRINS technique contributed to further the understanding of biological phenomena and describes the different possibilities and applications of the PRINS method in several biological and clinical fields (pre-implantation testing, prenatal, constitutional and oncologic genetic diagnosis).

Detection of the protozoan Neospora caninum Using in situ Polymerase Chain Reaction

A new method to detect the protozoan Neospora caninum using indirect in situ polymerase chain reaction (PCR) is described. In situ PCR combines the advantages of the extraordinarily high sensitivity and specificity of PCR and the in situ representation of immunohistochemical methods. We describe an indirect in situ PCR, whereby the amplified products were detected using a primed in situ (PRINS) reaction with hapten-labeled nucleotides and visualized using fluorochrome-labeled antibodies. This technique was carried out in both infected cell cultures and formalin fixed, paraffin embedded tissues. Clear signals were obtained in the N. caninum positive samples using in situ PCR, whereas control slides with Toxoplasma gondii infected tissues always yielded negative results.

Fast multicolor primed in situ protocol for chromosome identification in isolated cells may be used for human oocytes and polar bodies

Objective To present and evaluate the use of a new ultra-fast multicolor primed in situ (PRINS) procedure for karyotyping human oocytes and first polar bodies. Design In situ chromosomal identification on isolated cells, using combinations of specific primers for chromosomes 1, 7, 9, 16, and 18 and fluorescent nucleotides. Setting Sixteen unfertilized oocytes were obtained from women participating in an IVF program. Patient(s) Five patients undergoing an IVF-ET. Intervention(s) In vitro unfertilized oocytes were fixed on slides, and sequential PRINS reactions were performed on each preparation. Main outcome measure(s) Ultrarapid in situ identification of three or four chromosomes on oocyte and polar body chromosome spreads. Result(s) On the basis of the direct in situ mixing of the colors of fluorochromes (FITC, TRITC, Cascade Blue) that were incorporated in sequential PRINS reactions, this method allows rapid and efficient labeling of three or four individual chromosomes. Each PRINS reaction consists of a unique 4- to 6-minute step for both in situ annealing and elongation. The procedure can be combined with fluorescence in situ hybridization (FISH) reactions. Conclusion(s) By simplifying the multicolor PRINS procedure, this new protocol should facilitate the use and adaptation of PRINS to chromosome screening. This approach could be used in parallel or in combination with FISH for efficient aneuploidy assessment on isolated cells.

Mouse telomere analysis using an optimized primed in situ (PRINS) labeling technique.

Telomeres are chromosomal elements composed of variable numbers of a TTAGGG repeated DNA sequence required for genomic stability. Telomeric length is correlated with the number of copies of this repeated DNA sequence and is an important property relevant to telomeric function. Recently, it has been demonstrated that the length of the shortest telomere, not average telomeric length, is important for cell viability and chromosomal stability. Consequently, assays permitting assessment of telomeric length are important for the analysis of genomic instability disorders. The length of individual telomeres can be analyzed using the primed in situ (PRINS) labeling reaction, which produces a labeled copy of the telomeric DNA repeats in situ. In this study, we tested different variables to optimize the PRINS reaction to enable it to be applied to the detection of mouse telomeric DNA and the study of telomeric length. The specificity, efficiency and uniformity of staining were evaluated using digital fluorescence microscopy. Labeling efficiency is dependent upon the conditions used to denature the telomeric DNA and reaction duration. Staining uniformity is increased at higher annealing and elongation temperatures as well as when a fluorescently labeled nucleotide is incorporated during the elongation step. Our results also indicate that chromosomal background staining is observed when a fluorochrome-labeled nucleotide is used as opposed to a hapten-labeled nucleotide. From this study, we conclude that an optimized PRINS technique can be reliably employed to analyze mouse telomeres and, compared with the FISH (fluorescence in situ hybridization) technique, presents advantages including greater cost efficiency and reduced processing time. These advantages may encourage wider use of the PRINS technique for quantitative evaluation of the length of individual telomeres in situ.

Primed in situ nucleic Acid labeling combined with immunocytochemistry to simultaneously localize DNA and proteins in cells and chromosomes.

In the past decade, the primed in situ (PRINS) labeling technique has become an alternative to fluorescence in situ hybridization (ISH) for the localization of nucleic acid sequences in chromosome, cell, and tissue preparations (, , , , , , , ). The PRINS method is based on the rapid annealing of unlabeled primers (restriction fragment, PCR product, or oligonucleotide) to complementary target sequences in situ. These primers serve as initiation sites for in situ chain elongation using Taq DNA polymerase and labeled nucleotides. Incorporated fluorochrome-labeled nucleotides can be detected directly by fluorescence microscopy, whereas haptenized (e.g., biotin, digoxigenin, dinitrophenyl) nucleotides can be visualized by the additional application of fluorochrome- or enzyme-conjugated avidin or antibody molecules (,,,), followed by fluorescence microscopy or brightfeld visualization of enzyme reaction products. Particularly, rapidity, simplicity, and cost-effectiveness have made the PRINS technique a useful tool in cytogenetics and cell biology. Its detection sensitivity, however, seems to be limited to repetitive targets for a long time. Only recently, the combined use of multiple oligonucleotides for 1 locus together with tyramide signal amplification have shown the first reproducible results demonstrating single-copy gene detection by PRINS ().

Preparation and analysis of spermatocyte meiotic pachytene bivalents of pigs for gene mapping.

Well-spread meiotic pachytene bivalents were obtained by using the prolonged hypotonic treatment combined with high chloroform Carnory's fixative solution from cells of the testes of domestic pigs. Comparison in the division index and length of pachytene bivalents with metaphase chromosomes showed that those of the former are 5 times higher and 3.42(1.87-5.98) times longer than those of the latter. Comparative studies on chromomere maps of bivalents and mitotic chromosomal G-bands were conducted by using the chromosome 12 as a example. Sex vesicle and various shapes of synaptic sex chromosomes have been observed. Two-color PRimed IN Situ (PRINS) labeling has been conducted successfully on pachytene bivalents of pigs.

Rapid chromosome detection by PRINS in human sperm.

Primed in situ (PRINS) labeling may be used for direct estimation of disomy rate in human sperm. We combined the PRINS procedure with a rapid and efficient NaOH pretreatment allowing simultaneous decondensation and denaturation of sperm nuclei. This enabled double labeling of human sperm within a 2-hr time span. Chromosome-specific primers have been defined for most human chromosomes, including those most frequently involved in aneuploidy. Using the dual-color PRINS method, we estimated incidences of disomy for 15 autosomes and the sex chromosomes in sperm samples from 6 normal fertile men. The frequency of disomy ranged from 0.28% to 0.36%. There were no significant interchromosomal differences in disomy rate, but chromosome 21 displayed a higher incidence of nondisjunction than did the other chromosomes.

Use of primed in situ labeling (PRINS) for the detection of telomeric deletions associated with mental retardation

Rearrangements involving the telomeric regions of human chromosomes are often associated with mental retardation. These rearrangements, however, are difficult to detect using conventional cytogenetic techniques. We propose the use of primed in situ (PRINS) labeling as an alternative to fluorescence in situ hybridization because it is very fast, reproducible, and simple to perform. Sixty-five children with unexplained mental retardation were studied using PRINS technology; two of them were shown to have a telomeric deletion.

Classical and molecular cytogenetics of the zebrafish, Danio rerio (Cyprinidae, Cypriniformes): an overview.

The zebrafish, Danio rerio, has recently become the model system for the genetic analysis of vertebrate development. This paper reviews the advances in zebrafish cytogenetics, obtained through classical and molecular techniques, which will lead to the assignment of specific linkage groups to specific chromosome pairs in the zebrafish genome project. Several chromosome pairs of the 50-chromosome karyotype of D. rerio were differentially stained by classical staining techniques and additional information has been obtained by molecular cytogenetics. Indeed, the analysis of constitutive heterochromatin by C-banding and base-specific fluorochrome staining had suggested a differential composition of peri- and paracentromeric constitutive heterochromatin. The chromosome mapping of distinct AT- and GC-rich zebrafish satellite DNAs by means of PRINS (Primed in situ) and multicolor FISH (Fluorescence in situ Hybridization) has confirmed this hypothesis, which therefore provided the chromosome localization of 10% of the zebrafish genome. The analysis of nucleolus organizer regions (NORs) by silver staining and by FISH with 18S rDNA has also revealed the existence of variable and inactive NORs, in addition to those on the terminal regions of the long arms of the three NOR-bearing chromosome pairs. Other multicopy genes, such as minor ribosomal genes, or multicopy repeats, such as telomere specific sequences, have now been mapped on zebrafish chromosomes. The latest advancement in zebrafish molecular cytogenetics is the chromosome mapping of single locus genes. Single-copy genes from each of the 25 genetic linkage groups are now being mapped on zebrafish chromosomes by using PAC clones.

Use of PRINS for preconception screening of polar bodies for common aneuploidies

It has been shown that preconceptional screening for oocyte aneuploidies could help increase the pregnancy rate after in vitro fertilization (IVF), particularly in cases of advanced maternal age. The FISH (fluorescent in situ hybridization) technique is usually used to examine the first polar body (I-PB) for oocyte screening and so avoid fertilizing and transferring embryos from aneuploid oocytes. We have tested the feasibility of using another technique, the primed in situ (PRINS) reaction for this purpose. PRINS is a rapid, inexpensive method of labelling chromosomes. Chromosomes were labelled by in situ annealing with chromosome-specific oligonucleotide primers, followed by primer extension with labelled nucleotides using Taq DNA polymerase. A total of 183 PRINS reactions were performed with primers for chromosomes 13, 16, 18, 21 or X on 63 I-PBs removed from oocytes that failed to become fertilized during IVF. Each I-PB underwent three successive double-labelling reactions and intense signals were obtained in less than 40 min. Our data suggest that PRINS may be a useful alternative or a complement to FISH for detecting the main aneuploidies in all oocytes obtained after follicular puncture.

Use of PRINS for preconception screening of polar bodies for common aneuploidies

It has been shown that preconceptional screening for oocyte aneuploidies could help increase the pregnancy rate after in vitro fertilization (IVF), particularly in cases of advanced maternal age. The FISH (fluorescent in situ hybridization) technique is usually used to examine the first polar body (I-PB) for oocyte screening and so avoid fertilizing and transferring embryos from aneuploid oocytes. We have tested the feasibility of using another technique, the primed in situ (PRINS) reaction for this purpose. PRINS is a rapid, inexpensive method of labelling chromosomes. Chromosomes were labelled by in situ annealing with chromosome-specific oligonucleotide primers, followed by primer extension with labelled nucleotides using Taq DNA polymerase. A total of 183 PRINS reactions were performed with primers for chromosomes 13, 16, 18, 21 or X on 63 I-PBs removed from oocytes that failed to become fertilized during IVF. Each I-PB underwent three successive double-labelling reactions and intense signals were obtained in less than 40 min. Our data suggest that PRINS may be a useful alternative or a complement to FISH for detecting the main aneuploidies in all oocytes obtained after follicular puncture. Copyright © 2000 John Wiley & Sons, Ltd.

Interphasic Analysis of Aneuploidy in Cancer Cell Lines Using Primed In Situ Labeling

The primed in situ (PRINS) labeling technique has been adapted to chromosomal screening of interphasic tumoral cells. A panel of ten chromosome-specific α-satellite DNA primers was used to evaluate numerical chromosome abnormalities in two colon cancer cell lines (Caco-2 and HT-29) and in three of their subpopulations (PF11, TC7, and HT29-MTX). In each cell line, the copy number distibution for different chromosomes showed different patterns. The observation of significant variations in the chromosome constitutions between subpopulations derived from the same original tumor suggests the common occurrence of chromosome copy number heterogeneity in tumoral cell lines. This study demonstrates that the PRINS procedure offers a simple and reliable method for in situ chromosomal screening, which could be efficiently used for karyotypic analysis of tumoral cells.

Chromosomal localization of zebrafish AluI repeats by primed in situ (PRINS) labeling

Two zebrafish AluI repeats were localized in metaphase chromosomes by means of the primed in situ (PRINS) labeling technique, using oligonucleotide primers based on published sequences. An AT-rich, tandemly repeated, long AluI restriction fragment (RFAL1) labeled the (peri)centromeric regions of all chromosomes. The GC-rich short frag- ment (RFAS) was found to be localized in the paracentromeric regions of 17 chromosome pairs, which were mostly subtelocentric. The RFAS labeling pattern generally fits the previously described chromomycin A₃ (CMA₃) staining pattern. The differential composition of heterochromatin in zebrafish chromosomes is discussed.

Interleukin-4 enhances 15-lipoxygenase activity and incorporation of 15(S)-HETE into cellular phospholipids in cultured pulmonary epithelial cells.

15(S)-Hydroxyeicosatetraenoic acid (15[S]-HETE) is a 15-lipoxygenase (15-LO) metabolite that may play an important role in different pulmonary diseases. 15-HETE is synthesized by different epithelial cells and may be subsequently incorporated into cellular phospholipids. We studied the role of interleukin-4 (IL-4) on 15-LO activity and on 15(S)-HETE incorporation into cellular phospholipids by WI-26 pulmonary epithelial cells. 15-LO activity was evaluated by measuring 15(S)-HETE production, through combined reverse-phase-high-pressure liquid chromatography (RP-HPLC) separation and specific radioimmunoassay (RIA), after incubation with arachidonic acid (AA). We also studied 15-LO messenger RNA (mRNA) expression, using primed in situ (PRINS) labeling. IL-4 (10 ng/ml) markedly increased the percentage of 15-LO mRNA-bearing cells as well as 15-LO activity after 24, 48, and 72 h, with a maximal response at 48 h. Uptake and incorporation into cellular phospholipid was studied with [3H]15(S)-HETE, which showed that IL-4 was able to increase significantly 15(S)-HETE incorporation into WI-26 cells, with a maximal effect observed at 72 h. Cellular-lipid-associated [3H]15(S)-HETE, evaluated with RP-HPLC after base-catalyzed hydrolysis, increased concomitantly with disappearance of the radiolabel from the supernatant. Class separation of cellular lipids with normal-phase HPLC (NP-HPLC) showed that IL-4 increased [3H]15(S)- HETE incorporation mainly in the phosphatidylinositol (PI) fraction. The ability of IL-4 to promote 15-LO activity and incorporation into cellular phospholipids of human lung epithelial cells may be important in airway inflammation and in modulation of the potential autocrine function of 15(S)-HETE.

Fetal cells in maternal blood: the use of primed in situ (PRINS) labelling technique for fetal cell detection and sex assessment.

Prenatal diagnosis is presently performed following invasive procedures with variable risks of fetal loss; non-invasive procedures using fetal cells in maternal blood would be welcome for the early detection of fetal sex or aneuploidy. We describe a simple and rapid protocol to detect fetal cells and thus to assess fetal sex. In a first step, nucleated blood cells were separated into mononuclear and polynuclear cells using a double density gradient centrifugation. In a second step primed in situ (PRINS) labelling technique was performed to label Y-chromosomes. 15 samples were studied and correct gender assignment was made in 13/15. The number of labelled nuclei was higher in polynuclear cell phases than in mononuclear cell phases. Moreover, the polylobular aspect of labelled nuclei from polynuclear cell phases strongly suggested that they could belong to fetal polynuclear cells. The PRINS technique combines some advantages of FISH, such as visual assessment of in situ chromosome labelling and the powerful specificity and sensitivity of PCR. In association with a simple enrichment procedure it constitutes a rapid protocol for fetal cell detection, non-invasive early prenatal sex assessment, and could further be applied to detect the main viable aneuploidies.

Rapid characterization of human chromosomes in hybrid cell lines by primed in situ (PRINS) labeling.

The primed in situ (PRINS) labeling technique allows a rapid and specific labeling of human chromosomes in situ. This method is based on annealing of specific oligonucleotide primers and subsequent primer extension by a Taq DNA polymerase. We have developed a PRINS protocole for the cytogenetic analysis of somatic hybrid cell lines. Painting of human chromosomes is performed using Alu specific primers. Individual human chromosomes are identified using chromosome-specific alpha-satellite primers. The method was successfully tested to 3 different human-hamster hybrid cell lines. This approach provides an interesting alternative to classical cytogenetic and in situ hybridization techniques for the characterization of the human content of hybrid cell lines.