Normal Diploid Controls Research Articles

Generation and genetic analysis of a rainbow trout (Oncorhynchus mykiss) clonal line produced by gynogenesis

The efficiency of heat-shock treatments for producing gynogenetic double haploids was evaluated using a rainbow trout (Oncorhynchus mykiss) strain locally adapted in Brazil (2002 broodstock). Eleven and 15 females produced by conventional reproduction (FC) or meiotic gynogenesis (FG) were used as egg donors, respectively. Oocytes from each female were randomly divided into five uneven lots which were submitted to different treatments: (C2n) Normal diploid control, (H) Haploid control, (G-Me) Meiotic gynogenetic, (G-Mi) Mitotic gynogenetic, and (4 n) Control of suppression of the 1st cell cleavage. Homozygous yellow-colored (Dominant albino - DA) males were used as semen donors. Gynogenesis was induced by insemination of eggs with DA semen genetically inactivated by ultraviolet irradiation. Diploidization of gynogenic individuals was induced by an early heat shock to block 2nd polar body extrusion (G-Me), or a late heat shock to suppress first mitosis (G-Mi). Overall mean survival rates to eyed-egg and first feeding stages of treated groups, relative to respective C2n control group were: 19.21 and 14.90%, 13.02 and 8.09%, and 29.94 and 16.48%, for treatments G-Me, G-Mi and 4 n, respectively. When compared with C2n, all treatments showed lower survival rates at both stages. Survival of G-Me and G-Mi progeny from FG females were significantly higher when compared to progeny from FC females for both stages. Progenies from two different females observed to be 100% homozygous in analyzed informative microsatellite markers were used in subsequent cycles of gynogenic reproduction. One line originally derived from a single gynogenic female was reproduced for nine successive generations of meiotic gynogenetic reproduction. Forty animals from the ninth successive generation of gynogenetic reproduction were analyzed with a low-density SNP (Single Nucleotide Polymorphism) panel. All individuals were observed to be homozygous for identical alleles at all SNPs, therefore indicating that the established mitotic gynogenetic line is composed of 100% clonal individuals.

Characterization of parent-of-origin methylation using the Illumina Infinium MethylationEPIC array platform.

This study aimed to establish a catalog of probes corresponding to imprinted differentially methylated regions (DMRs) on the Infinium HumanMethylationEPIC BeadChip. Reciprocal uniparental diploidies with low normal biparental mosaic contribution, together with normal diploid controls, were subjected to EPIC BeadChip hybridization. The methylation profiles were assessed for imprinted differential methylation. Top candidates were validated using locus-specific PCR-based assays. Seven hundred and eighty-nine CpG probes coincided with 50 known imprinted DMRs and 467CpG probes corresponding to 124 novel imprinted DMR candidates were identified. Validation led to identification of several subtle DMRs within known imprinted domains as well as novel maternally methylated regions associated with PTCHD3 and JAKMIP1. Our comprehensive list of bona fide-imprinted DMR probes will simplify and facilitate methylation profiling of individuals with imprinting disorders and is applicable to other diseases in which aberrant imprinting has been implicated, such as cancer and fetal growth.

Disruption of Direct 3-Dimensional (3D) Telomere-TRF2 (Telomere Related Factor 2) Interaction Is a Hallmark of Primary Hodgkin (H) and Reed-Sternberg (RS) Cells

Introduction: Numerical and complex structural chromosomal aberrations are regularly identified in primary mononuclear H and multinucleated RS cells and in Hodgkin's lymphoma (HL) derived H cell lines. The transition from H to RS cells is associated with dynamic progressive 3D telomere dysfunction, major changes in the telomere-protecting shelterin complex and formation of giant "zebra" chromosomes. Analogous findings are observed in an in vitro model for post-germinal center B-cell, Epstein-Barr-virus (EBV)-associated HL (Blood. 2015;125 (13):2101-2110). In this experimental system the EBV encoded oncogene LMP1 mediates multinuclearity through downregulation of TRF2. Thus, we hypothesized that the 3D interaction of telomeres and TRF2 is directly associated with the formation of H and RS cells. To this end, we developed a combined quantitative 3D TRF2-telomere immuno FISH protocol (3D TRF2/Telo-Q-FISH) and applied it to monolayers of primary H and RS cells enriched from diagnostic lymph node biopsies allowing 3D analysis of the entire nuclear content (often not achieved using laser microdissection of H and RS cells, given that their nuclei are generally >10 um in diameter and that this technique is performed on 5 um sections).Methods: 3D TRF2/Telo-Q-FISH(Blood. 2015;125 (13):2101-2110; data supplement) was performed on cultured BJ-5ta normal diploid fibroblasts and B-cell rich suspensions (negative selection) of diagnostic lymph nodes, cultured for 12-36 hours. Quality of H and RS cells was assessed by Wright-Giemsa staining. Lymphocytes, H and RS cells of 10 classical HL were analyzed, 3 of them containing LMP1+ tumor cells (EBV-associated HL).Results: Whereas the BJ-5ta normal diploid control fibroblasts and most internal control lymphocytes showed quantitatively and qualitatively intact 1:1, tight physical 3D association of TRF2 with telomeres, with only few very short telomeres (<20%), the H and RS cells of all 10 cases consistently displayed telomere-TRF2 steric disruption, with a significant increase of very short telomeres (49-71%). Interestingly, two opposite disruption patterns of the 3D telomere-TRF2 interaction were identified.The 3 LMP1+ cases were characterized by abundant telomeres without TRF2 interaction (de-protected). TRF2 spots were rare and the quantitative ratio of telomere/TRF2 signals was > 6:1. This loss of TRF2 expression showed gradual progression from H to RS cells.The 7 LMP1- cases showed a different 3D telomere-TRF2 pattern. Three cases with clinically aggressive disease (stages IVA, IVB and IIIB) were characterized by abundant TRF2 signals, most of them not associated with telomeres. In two of them huge multinucleated RS "ghost" cells without any or only few telomeres but numerous TRF2 signals and internuclear DNA-bridges were observed. In one of these two and in the third case bi-nucleated RS cells with grossly different nuclear telomere content of nucleus one and nucleus two were present. Most importantly, these 3 cases showed an inversion of the quantitative ratio of telomere/TRF2 signals <1:3 and the progressive loss of telomere signals from H to RS cells. The remaining 4 cases had a mixed pattern with an overall moderate loss of TRF2 signals including TRF2 de-protected telomeres but also TRF2 signals not associated with telomeres (quantitative, telomere/TRF2 signals ratio of about 1.5-2:1).Conclusions: Disruption of the 3D TRF2-telomere interaction was demonstrated in 100% of primary H and RS cells. However, two opposite pathogenetic mechanisms appear to be operative: in EBV-associated HL telomere de-protection is due to an impressive loss of TRF2 signals physically linked to telomeres, in agreement with the hypothesis of our in vitro model for EBV-associated HL. In clinically aggressive EBV-negative HL massive attrition of telomere signals is associated with an overwhelming increase in TRF2 signals that are not associated with telomeres. In both scenarios, the telomeres are predominantly very short and the crucial 3D interaction is disrupted, thereby qualifying HL as a shelterin-associated disease with the telomere-shelterin complex playing the role of a "plaque tournante" in the pathogenesis of HL. Further molecular dissection of the shelterin-telomere interaction may offer new therapeutic targets for refractory/relapsing disease. DisclosuresNo relevant conflicts of interest to declare.

Abstract LB-122: High-throughput tumor genomic profiling by massively parallel sequencing

Abstract The use of tumor genetic and molecular information to accurately predict a patient's response to therapy is critical to personalized cancer medicine. Knowledge of somatic genetic alterations in tumors - including mutations, copy number alterations, and pharmacogenomic polymorphisms - should ultimately facilitate individualized approaches to cancer treatment. However, the development and real-world implementation of comprehensive genomic profiling has not yet been achieved. Here, we describe the development of a high-throughput, massively parallel sequencing platform to detect tumor genomic alterations that may provide an efficient and cost-effective means to address this challenge. Our approach combines several recent innovations - solution phase hybrid selection, DNA barcoding and pooling, and massively parallel sequencing - to achieve a high-throughput genomic profiling platform. We designed and synthesized ∼7000 biotinylated RNA baits corresponding to the coding sequence of the top 150 “druggable” or potentially “actionable” genes known to undergo somatic genomic alterations in cancer. We obtained genomic DNA from cell lines with known genetic alterations and used this DNA to generate barcoded sequencing libraries. Following quantification of libraries, equimolar pools were generated consisting of up to 12 barcoded tumor DNAs and normal diploid control DNAs. These DNA pools were subjected to solution-phase hybrid capture with the biotinylated RNA baits followed by massively parallel sequencing. The sequencing data were deconvoluted to match all high-quality reads with the corresponding tumor samples and call base mutations and copy number alterations. In all samples tested, sequence reads were highly specific to the targeted exonic regions. In addition to detecting mutations, exon capture detected high-level amplifications and deletions, two types of “actionable” genomic alterations that are poorly detectable by mass spectrometric approaches to mutation profiling. Comparison with copy number data previously obtained using high-density SNP array data demonstrated a robust correlation. As a proof-of-principle, we performed the entire profiling approach on clinical tumor specimens from patients with refractory, aggressive cancers to assess the use of genomic profiling in a real-world situation where knowledge of “driver” genetic alterations might prove valuable. Altogether, our data endorse this sequencing-based approach as a promising method to detect critical “actionable” genetic alterations in a large panel of cancer genes. The platform is scalable, refractory to contaminating stromal DNA or hyperploidy, and completed at a fraction of the cost of comparable sequencing approaches. By providing a rapid, sensitive, cost-effective means to sequence &amp;gt;100 cancer genes deeply and simultaneously, this approach may ultimately empower the rational selection of specific drugs targeting the genetic alterations in each patient's tumor - clearly an unmet need in oncology. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-122.

Karyotypes of Diploid and Triploid Mercenaria mercenaria (Linnaeus)

Northern quahogs (=hard clam; Mercenaria mercenaria) is a commercially important bivalve naturally distributed along the American Atlantic coast. Because the hard clam was first introduced into China as a candidate for aquaculture in 1997, research has been conducted on its ecologic habits, ecological physiology, metabolism, growth, reproduction, artificial propagation, and seed culture. Little information about their chromosomes and karyotype is available. The present study is designed to study the karyotype of the diploids and triploids to provide a reference to genetic research on introduced M. mercenaria. Triploids were produced by inhibiting the release of second polar body (PBII) with a treatment of 450 μmol/mL 6-deimethylaminopurine (6-DMAP), with untreated fertilized eggs developing as normal diploid controls. Chromosome preparations were made from trochophore larvae with drop-splash method. Chromosomes were measured and classified according to Leven criteria. Statistical analysis of relative length and arm ratio revealed that, this species had a normal diploid chromosome number of 2n = 38, and the karyotype consists of 15 pairs of metacentric (M) and 4 pairs of submetacentric (SM) chromosomes, NF = 76. The triploid karyotype formula was 45 M 12 SM, NF = 114. The heterochromosomes and satellite chromosomes were not observed in all chromosomes in the complement.

Induction of diploid gynogenesis in southern flounder ( Paralichthys lethostigma) with homologous and heterologous sperm

Effective methods for induction of diploid gynogenesis in North American flounder of the genus Paralichthys are needed to initiate monosex culture, which will allow growers to take advantage of the more rapid growth and larger size attained by females. To test methods for inducing diploid gynogenesis in southern flounder ( Paralichthys lethostigma) using homologous sperm, four treatments, named for their expected outcome, were employed: haploid, diploid, triploid, and gynogenetic diploid. Diploid gynogenesis was induced by activating egg development with UV-irradiated flounder sperm (70 J/cm 2) for 3–4 min in seawater, and then subjecting the eggs to cold shock in 0–2 °C seawater for 45–50 min. Cold shock was used to prevent extrusion of the second polar body. Control treatments omitted one or more of these steps to separately assess the effectiveness of UV irradiation and cold shock. Larvae were observed for physical abnormalities and then histologically processed for ploidy determination. Haploid larvae exhibited abnormal external morphology while diploid, gynogenetic diploid, and triploid larvae showed normal morphologies. Cross-sectional areas of erythrocyte nuclei were measured for larvae in each treatment group and significant differences were found. Nuclear areas for treatment groups corresponded to predicted ploidy (triploid>diploid>haploid) and did not differ between normal diploid controls and gynogenetic diploids. These results suggest that the procedures of sperm irradiation and egg cold shock successfully generated gynogenetic diploids. Due to the low volumes of semen produced by male flounder, and to eliminate any potential genetic contribution by homologous sperm, activation of flounder eggs with heterologous sperm was also investigated. Induction of diploid gynogenesis was successful when flounder eggs were fertilized with irradiated (50 J/cm 2) sperm from striped mullet ( Mugil cephalus), and then cold shocked. This work provides procedures for induction of diploid gynogenesis in southern flounder using homologous and heterologous sperm, and validates a method for verification of ploidy in larval fish.

An efficient method for blocking the 1st mitotic cleavage of fish zygote using combined thermal treatment, exemplified by mud loach ( Misgurnus mizolepis)

Blocking the first mitotic cleavage of the zygote is a key tool for chromosome-set manipulations in fish. We developed an improved method for inducing tetraploidy by blocking the mitosis with a combination of heat shock at 40.5 °C for 1, 2, or 3 min followed by cold shock at 1.5 °C for 30, 45, or 60 min. When applied during the first cleavage metaphase of mud loach ( Misgurnus mizolepis) zygotes, the optimal combination was heat for 2 min followed by cold for 45 min. At 1 month, the frequency of 4N survivors and the yield from total eggs fertilized was 55.7 and 14.4%, respectively, compared to heat shock alone with 20.0% efficiency and 3.6% yield. The effectiveness of the procedure was confirmed by diploid mitotic gynogenesis using transgenic markers. The overall yield of homozygous diploids, 34.0%, was better than that for single heat shock, 17.3%. The tetraploids and homozygous diploids had higher early mortality than normal diploid controls. However, at 1 month, the viability of the tetraploids was the same as normal diploids. For gynogenetic diploids, the survival was similar to normal diploids after 3 months. The high efficiency of this new protocol extends the opportunity to study polyploidy in basic and applied research.

Sex determination and polyploid gigantism in the dwarf surfclam (Mulinia lateralis Say).

Mulinia lateralis, the dwarf surfclam, is a suitable model for bivalve genetics because it is hardy and has a short generation time. In this study, gynogenetic and triploid M. lateralis were successfully induced. For gynogenesis, eggs were fertilized with sperm irradiated with ultraviolet light and subsequently treated with cytochalasin B to block the release of the second polar body (PB2). Triploidy was induced by blocking PB2 in normally fertilized eggs. The survival of gynogenetic diploids was very low, only 0.7% to 8 days post-fertilization (PF), compared with 15.2% in the triploid groups and 27.5% in the normal diploid control. Larvae in all groups metamorphosed at 8-10 days PF, and there was no significant post-larval mortality. At sexual maturation (2-3 months PF), all gynogenetic diploids were female, and there was no significant difference (P > 0.05) in sex ratio between diploids and triploids. These results suggested that the dwarf surfclam may have an XX-female, XY-male sex determination with Y-domination. Compared with diploids, triploids had a relative fecundity of 59% for females and 80% for males. Eggs produced by triploid females were 53% larger (P < 0.001) in volume than those from diploid females. In both length and weight measurements at three months PF, the gynogenetic diploids were not significantly (P > 0.33) different from normal diploid females, suggesting that inbreeding depression was minimal in meiosis II gynogens. Triploid clams were significantly larger (P < 0.001) than normal diploids. We hypothesize that the increased body-size in triploids was caused by a polyploid gigantism due to the increased cell volume and a lack of cell-number compensation.

Chromosomal aberrations in irradiated Down's syndrome fibroblasts

We have compared the frequencies of chromosomal aberrations in skin fibroblasts from persons with Down's syndrome (trisomy 21) with those from normal diploid controls exposed to 60Co γ-radiation in vitro. No difference between the chromosomal radiosensitivities of the two groups was observed, nor did the two groups differ in the background frequency of spontaneous aberrations. These data support the hypothesis that the increased in vitro chromosomal radiosensitivity of lymphocytes reported to be associated with trisomy 21 is not typical of all tissues.

Survival, growth and fertility of gynogenetic diploids induced in the cyprinid loach, Misgurnus anguillicaudatus

Gynogenetic diploids were induced in the loach, Misgurnus anguillicaudatus, by applying cold shock to the egg after fertilization with spermatozoa genetically inactivated by ultraviolet-ray irradiation, to examine their survival, growth and fertility. Eggs were obtained from orange phenotype loach (homozygous for a recessive demelanogenesis gene). Spermatozoa were obtained from black phenotype loach (homozygous for a dominant melanogenesis gene), and from common carp, Cyprinus carpio. All hybrids between loach and carp were inviably abnormal. Eggs fertilized with loach or carp spermatozoa irradiated at doses of 6000 and 12000 erg/mm 2 and then treated with cold water (1°C for one hour 4 and 5 min after fertilization developed into embryos with 61–78% survival. About 70–87% of newly-hatched fry were normal in appearance. Gynogenetic fish induced by both loach and carp spermatozoa showed lower rates of survival and growth than did the normal diploid controls. All gynogenetic fish from carp spermatozoa were female and 94.9% of the gynogenetic fish from loach spermatozoa were female. The second gynogenetic offsprings were produced from females selected from both gynogenetic groups.

Frequencies of Sister Chromatid Exchanges in Heteroploid Cell Lines of Human Melanoma Origin&lt;xref ref-type="fn" rid="fn2"&gt;2&lt;/xref&gt;

Sister chromatid exchange(s) (SCE) per cell and chromosome in five heteroploid cell lines and sublines of human melanoma origin were distinctly more numerous than those in the normal diploid control. The SCE per unit chromosome length was higher in stable heteroploid C32 (0.041-0.047) and C9 (0.056) than in normal diploid control. The SCE per unit chromosome length was higher in stable heteroploid C32 (0.041-0.047) and C9 (0.056) than in normal diploid control C182 (0.036), which suggested that the longer the total chromosome length of the cell genome, the higher the SCE per cell as well as per unit chromosome length. These malignant heteroploid lines had karyotypes ranging from hypodiploidy to hypotriploidy and were extremely heterogeneous in one cell line but unusually homogenous and stable in others. However, none of these properties could be correlated to the increase in SCE frequency. As compared to C32-r7, the subline C32-RO had more SCE per cell, chromosome, and unit chromosome length for the genome as well as for 9 of 11 individual chromosomes and chromosome groups. In contrast, the C32-RO specific ring marker chromosome had only 40% of the SCE of its nonring homologue in C32-r7 cells. Interestingly, C32-RO and its ring marker were less stable than C32-r7 and the nonring marker during in vitro growth. There was no noticeable difference between C32-r7 and its derivative C32-r7-nu-1, which implied that transplantation into nude mice did not alter the expression of SCE in this subline.

Establishment of normal diploid and malignant heteroploid cell lines from non-treated and benzo(a)pyrene treated hamster embryo cell cultures.

Two normal diploid control cell lines and a heteroploid malignant transformed cell line from B(a)P treated hamster embryo cell cultures were established. The 14-month-old B(a)P transformed cell line grew 8-times faster than the 20-month-old control cell line. The control cell line showed normal diploid chromosome complement in 93% cells and heteroploidy in 7% cells while B(a)P treated line showed 83% heteroploid cells and only 17% diploid cells. This is the first report on the establishment of diploid hamster cell cultures grown for extended period.

A long-term effect of radiation on chromosomes of cultured human fibroblasts

Six monolayer fibroblast tissue cultures of different human origin were used for this study. Portions of each culture were Co 60-irradiated with varying doses of up to 500 rad at various times in their life history. The cultures were then maintained and observed for at least 10 months along with their normanl unirradiated controls for long-term changes in their chromosome composition. At least one of the irradiated fractions of each of the six primary cultures ultimately became pseudodiploid with one or more conspicuous markers, while the corresponding unirradiated control cultures remained diploid. In those cultures which provided favorable material for sequential observations after marker appearance, the pseudodiploid cells with the marker chromosomes soon prevailed in the culture. None of the irradiated cultures showed signs of alteration into established cell lines as of this writing. They all appear to have stopped rapid growth at approximately the same time as their normal diploid controls.

Continuous cultivation of cells derived from haploid Rana pipiens embryos

A technic is described which permits the in vitro cultivation of cells derived from advanced androgenetic haploid embryos of the frog Rana pipiens, or from normal diploid controls. Monolayer cell cultures are obtained which may be repeatedly subcultured by trypsinization, and which grow as sheets and clumps of cells of fibroblastic morphology. Study of the chromosomes of dividing cells in the cultures shows that a minority of diploid cells (five per cent or less) occur in the early subculture generations. These increase, with succeeding transfers, relative to the haploid population, until eventually they become the majority type. Such diploidized cultures of haploid origin do not, however, grow at the same rate as do the cultures of normal diploid origin. This is taken to suggest the origin of the diploidized cells by an endomitotic process.