Biolistic transformation of haploid isolated microspores of barley (Hordeum vulgare L.)

Genome ◽  
1997 ◽  
Vol 40 (4) ◽  
pp. 570-581 ◽  
Author(s):  
Qing A. Yao ◽  
Ecaterina Simion ◽  
Manilal William ◽  
Joan Krochko ◽  
Ken J. Kasha
Keyword(s):  
Transgenic Plants ◽  
Southern Blot ◽  
Blot Analysis ◽  
Southern Blot Analysis ◽  
Nuclear Dna ◽  
Active Agent ◽  
Biolistic Transformation ◽  
Pcr Analysis ◽  
Bar Gene ◽  
Isolated Microspores

Transgenic barley plants were produced by the direct delivery of plasmid DNA into isolated microspores of barley cv. Igri using high velocity microprojectiles. The plasmid pAHC25 contained the uidA and bar genes, each under the control of a maize Ubi1 promoter. Bombarded microspores were cultured and selected on solid medium containing varying concentrations (2–5 mg/L) of the Basta herbicide active agent bialaphos. The effectiveness of selection with bialaphos depended on its interaction with the medium component glutamine. Six transgenic plants (R0) were obtained, and the presence of the uidA and bar genes and their integration into nuclear DNA in transformed R0 plants were confirmed by PCR and Southern blot analysis. Phosphinothricin acetyltransferase activity was observed in all six R0 transgenic plants, whereas none showed β-glucuronidase (GUS) activity in histochemical GUS assays. Two of the six R0 plants were haploid and sterile; one of them was trisomic and partially sterile; the remainder were diploid, but one of them was also sterile. Inheritance of the transgenes in progeny of three seed-producing transgenic plants was investigated. Southern blot analysis of genomic DNA from R1 plants showed that the introduced bar and uidA genes were hemizygous and stably cotransmitted to the R1 progeny derived from self-pollination. Analysis of Basta resistance and the integration of the bar gene by PCR analysis in R1 plants indicated that the bar gene was being inherited and expressed as a single dominant trait. Fluorescent in situ hybridization was performed on chromosomes of the trisomic plant to confirm the presence of transgenes in the genome.Key words: barley, microspore, biolistic transformation, bialaphos, haploid, FISH.

Detection of single-copy genes in DNA from transgenic plants by nonradioactive Southern blot analysis

1997 ◽  
Vol 7 (1) ◽  
pp. 79-84 ◽  
Author(s):  
Matthew S. McCabe ◽  
J. Brian Power ◽  
Ad M. M. de Laat ◽  
Michael R. Davey
Keyword(s):  
Transgenic Plants ◽  
Southern Blot ◽  
Blot Analysis ◽  
Southern Blot Analysis ◽  
Single Copy

Detection of Clonal IGH Gene Rearrangements: Summary of Molecular Oncology Surveys of the College of American Pathologists

2007 ◽  
Vol 131 (2) ◽  
pp. 185-189
Author(s):  
Marina N. Nikiforova ◽  
Eric D. Hsi ◽  
Rita M. Braziel ◽  
Margaret L. Gulley ◽  
Debra G. B. Leonard ◽  
...  
Keyword(s):  
Southern Blot ◽  
Blot Analysis ◽  
Southern Blot Analysis ◽  
Response Rate ◽  
Pcr Amplification ◽  
Pcr Analysis ◽  
Gene Rearrangements ◽  
Laboratory Practice ◽  
Molecular Oncology ◽  
Fresh Frozen

Abstract Context.—The diagnosis of B-cell lymphoid malignancy can frequently be substantiated by detecting clonal immunoglobulin heavy chain (IGH) gene rearrangements, which is typically done by polymerase chain reaction (PCR) amplification and/or Southern blot analysis. Objective.—To characterize current laboratory practice for the assessment of IGH rearrangements and to identify opportunities for improvement. Design.—The data from the Molecular Oncology Proficiency Survey distributed to participating laboratories by the Molecular Pathology Committee of the College of American Pathologists from 1998 through 2003 were analyzed. Results.—Thirty-nine proficiency survey specimens (29 positive and 10 negative for clonal IGH rearrangements) were distributed. For Southern blot analysis, 944 results were reported, with a successful response rate of 95%. For PCR detection, 2349 results were reported, with a successful response rate of 72%. A higher rate of successful responses by PCR was achieved using framework 3 primers in combination with other frameworks (82%) compared with framework 3 primers only (76%) and when fresh/ frozen (72%) compared with paraffin-embedded (65%) tissues were analyzed. Conclusions.—The performance of the participating laboratories was very good, by both Southern blot and PCR analysis. As expected, Southern blot analysis consistently detects a higher proportion of IGH rearrangements than PCR analysis. Further improvement and standardization of the IGH PCR assay is important if it is to replace Southern blot analysis as the standard method. Participation in this survey is a valuable tool for assessing laboratory performance and it directs our attention to areas where we may improve laboratory practice.

Value of molecular monitoring during the treatment of chronic myeloid leukemia: a Cancer and Leukemia Group B study.

1997 ◽  
Vol 15 (1) ◽  
pp. 26-36 ◽  
Author(s):  
W Stock ◽  
C A Westbrook ◽  
B Peterson ◽  
D C Arthur ◽  
T P Szatrowski ◽  
...  
Keyword(s):  
Southern Blot ◽  
Blot Analysis ◽  
Cytogenetic Analysis ◽  
Southern Blot Analysis ◽  
Residual Disease ◽  
Philadelphia Chromosome ◽  
Chronic Phase ◽  
Pcr Analysis ◽  
Molecular Monitoring ◽  
Blood Samples

PURPOSE Disappearance of the Philadelphia chromosome during treatment for chronic myeloid leukemia (CML) has become an important therapeutic end point. To determine the additional value of molecular monitoring during treatment for CML, we performed a prospective, sequential analysis using quantitative Southern blot monitoring of BCR gene rearrangements of blood and marrow samples from Cancer and Leukemia Group B (CALGB) study 8761. PATIENTS AND METHODS Sixty-four previously untreated adults with chronic-phase CML who were enrolled onto CALGB 8761, a molecular-monitoring companion study to a treatment study for adults with chronic-phase CML (CALGB 9013). Treatment consisted of repetitive cycles of interferon alfa and low-dose subcutaneous cytarabine. Blood and marrow Southern blot quantitation of BCR gene rearrangements was compared with marrow cytogenetic analysis before the initiation of treatment and of specified points during therapy. Reverse-transcriptase polymerase chain reaction (RT-PCR) analysis was performed to detect residual disease in patients who achieved a complete response by Southern blot or cytogenetic analysis. RESULTS Quantitative molecular monitoring by Southern blot analysis of blood samples was found to be equivalent to marrow monitoring at all time points. Twelve of 62 (19%) follow-up samples studied by Southern blot analysis had a complete loss of BCR gene rearrangement in matched marrow and blood specimens. Southern blot monitoring of blood samples was also found to be highly correlated to marrow cytogenetic evaluation at all points, although there were four discordant cases in which Southern blot analysis of blood showed no BCR gene rearrangement, yet demonstrated from 12% to 20% Philadelphia chromosome-positive metaphase cells in the marrow. RT-PCR analysis detected residual disease in five of six patients in whom no malignant cells were detected using Southern blot or cytogenetic analyses. CONCLUSION Quantitative Southern blot analysis of blood samples may be substituted for bone marrow to monitor the response to therapy in CML and results in the need for fewer bone marrow examinations. To avoid overestimating the degree of response, marrow cytogenetic analysis should be performed when patients achieve a complete response by Southern blot monitoring. This approach provides a rational, cost-effective strategy to monitor the effect of treatment of individual patients, as well as to analyze large clinical trials in CML.

scholarly journals Long-term engraftment of normal and post-5-fluorouracil murine marrow into normal nonmyeloablated mice [see comments]

Blood ◽  
1993 ◽  
Vol 81 (10) ◽  
pp. 2566-2571 ◽  
Author(s):  
FM Stewart ◽  
RB Crittenden ◽  
PA Lowry ◽  
S Pearson-White ◽  
PJ Quesenberry
Keyword(s):  
Southern Blot ◽  
Blot Analysis ◽  
Southern Blot Analysis ◽  
Normal Male ◽  
Normal Donor ◽  
Long Term Stability ◽  
Donor Bone Marrow ◽  
Male Donor ◽  
Marrow Cells

We report the successful long-term engraftment of normal male donor bone marrow (BM) transfused into noncytoablated female mice, challenging the assumption that “niches” need to be created for marrow to engraft. We have used chromosomal banding and Southern blot analysis to identify transplanted male marrow cells, and shown the long-term stability of the chimeric marrows. Balb/C, BDF1, or CBA-J female hosts (no irradiation) received for 5 consecutive days 40 x 10(6) male cells (per day) of the same strain, and repopulation patterns were observed. Parallel studies were performed using tibia/femur equivalents of normal marrow or marrow from Balb/C mice pretreated 6 days previously with 150 mg/kg 5-fluorouracil (5-FU). Chromosome banding techniques showed that 5% to 46% of marrow cells were male 3 to 9 months posttransplant with normal donor marrow. Southern blot analysis, using the pY2 probe, showed continued engraftment at 21 to 25 months posttransplant, ranging from 15% to 42% male engrafted cells in marrow. Normal donor male marrow engrafted significantly better than 5-FU-pretreated male marrow as shown 1 to 12 months posttransplant in non-cytoablated female recipients. Percentages of male engrafted cells in BM ranged from 23% to 78% for recipients of normal donor marrow and from 0.1% to 39% for recipients of 5-FU marrow. Mean engraftment for 6 mice receiving normal marrow was 38%, whereas that for 6 mice receiving post-5-FU marrow was 8%, as assayed 1 to 3 months posttransplant. At 10 to 12 months, mean engraftment for the normal donor group was 46%, compared with 16% for the 5-FU group. The patterns of engraftment with normal and 5-FU marrow were similar for spleen and thymus. These results show that long-term chimerism can be established after transplantation of normal donor marrow to normal nonirradiated host mice and indicate that marrow spaces do not have to be created for successful engraftment. They suggest that transplanted marrow competes equally with host marrow for marrow space. Finally, these data show that post-5-FU Balb/C male marrow is markedly inferior in the repopulation of Balb/C female host marrow, spleen, and thymus, and suggest that this population of cells may not be the ideal population for gene transfer studies.

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