Project Documentation & Protocols: Maize Gene Discovery Project: RescueMu Library Plates: FAQs
Contents: Index | Progress | Ordering | Screening | Sequences | Phenotypes | Protocols | FAQs
Grid Organization: Maize DNA is prepared from grids of transgenic plants containing transposed copies of RescueMu by pooling tissue samples along rows and along columns of the grid. One library is prepared for each row and for each column; a "full grid" contains 48 rows and 48 columns and generates 96 libraries. Some grids are smaller (as listed in the first column of the table). Click here for more information about grid organization.
Seed from Grid Plants: Each field plant is self-pollinated, if
possible, and the seed are sent to the Maize Coop. For plants that are male or female sterile, the plant is outcrossed with an anthocyanin tester; the outcross seed are grown at the Coop for self and sib-pollination to generate homozygotes. You will need an APHIS permit for use of transgenic corn and for interstate shipment of transgenic corn to obtain seed from the Coop.
Plasmid Types. The rescued plasmids (4.7 kb RescueMu, 2.9 kb MidiMu, 2.2 kb MiniMu) are a mixture of germinal and somatic insertion events. Somatic insertions are typically present in just a few cells in the plant and will be present in low abundance in just one row or one column library. Germinal insertions are much more abundant and should be found in one row and in one column library; the row and column numbers specify which plant contained the insertion. Pre-meiotic germinal insertions can be present in several rows or columns.
Plasmid Characteristics: Singly digested maize DNA is yielding plasmids with 3 - 25 kb of genomic DNA plus the RescueMu element, for a total size range of ~8 to 30 kb. RescueMu contains an ampicillin/carbenicillin resistance gene. MiniMu and MidiMu contain a kanamycin resistance gene. Consult the "Media" column of the table to verify media composition for each library plate.
Library Plate Production: Grids completed in summer 2000 and winter 2000-2001 are in production now. Consult the Table for availability dates; the Table will be updated as library plates are available.
RescueMu Sequencing Protocol: The same total DNA prep used for library production is double-digested with two restriction enzymes to make short insert templates for DNA sequencing. These libraries are screened to remove nearly all of the parental launching pad insertion plasmids, thus enriching for new insertion events. Using primers unique to the right or to the left terminal inverted repeat of RescueMu, we generate sequence from each side of most RescueMu insertion sites. The 9 bp host sequence duplication created by Mu insertion events plus the sequences at the ends of the TIRs are used to align the right and left sequences. In scanning the sequence outward from the TIRs, we note where restriction sites used in template preparation are located; sequence at these sites is "ambiguous" as it could be from the other side of the gene. The display of maize genomic DNA sequence generated in this protocol at ZmDB will take the ambiguity into account. GenBank deposits (gss database) are divided into four entries:
1. sequence from the right TIR up to the first "restriction site from the cloning protocol,
2. sequence after the right side restriction site,
3. sequence from the left TIR up to the restriction site in the cloning protocol,
4. left side sequence after this restriction site.
Somatic and Germinal Plasmids Contribute to Genomic Sequencing: Because Mu elements insert preferentially into genes, our sequencing effort is highly enriched for genes. Both somatic and germinal insertion plasmids yield genomic sequence important in gene discovery. We expect many genes not yet defined by an EST to be partially sequenced using the Mu plasmid rescue strategy. We are still fine-tuning the sampling procedure; currently we are attempting sequencing of 192 - 288 plasmids per row so that we recover germinal insertions 1 - 4 times and somatic insertions just once. We are experimenting with shallower sampling for greater efficiency in genomic sequencing.
RescueMu Sequencing Schedule: For the first half of 2001, Bret Schneider at the Stanford Genome & Technology Center is conducting all of the sequencing, while Brian Nakao and Khaled Sarsour concentrate on ESTs. Grid G (46 rows plus 2 columns as a cross-check) will require ~18 weeks to sequence.
Bioinformatics: All sequences are deposited immediately in GenBank. ZmDB will assemble the maize genomic DNA along with the EST collection.
What happened to grids A-E? Grid F? The tagging populations for grids A-E were segregating for the original RescueMu transgene arrays and multiple copies of the Basta-resistance co-transformation plasmid. These plants thus contain many copies of plasmids with carb/amp resistance markers. The efficiency of recovery of transposed RescueMu is low compared to recovery of the unwanted plasmid types. We are concentrating on preparing library plates and sequencing templates from our optimized grids from plants that contain only transposed RescueMu elements. Terrible weather conditions compromised the seed yield from selfed grid F plants, and preparation of library plates from this grid has been postponed until seed increases are available.
Understanding the organization of grids to order the correct seed: In grids G, H, I, K, and P the plant numbers in the field correspond exactly to the pooled row and column samples.
Example: Plant K row 5 - column 17 generated two leaf samples: one for the pool of row 5 and one for the pool of column 17. If you use PCR to screen a K library plate and got positive results for column 17 and row 5, then you would order seed from Coop for K5-17.
Grid M is an exception and you need to consult the chart posted below. For this grid, the pools of row and column materials were made only from plants that had a good selfed ear. As a consequence the seed designation is the intersection of the "row" and "column" information from RescueMu sequencing or your own PCR.
Example: Positive PCR results for row 5 and column 1 results in the intersection for M4-46 and this is the stock that you want.
Getting an APHIS permit for interstate shipment of RescueMu transgenic seed is easier than ever. New rules require APHIS to issue the permit within 30 days of receipt or contact you for clarification. APHIS has requested shorter and shorter descriptions of materials as well.
Multiple RescueMu permits have been issued, and APHIS and a number of the states are now used to the applications. The category for the transgenic seed is 00, and the descriptor title for the experiment is "transposon tagging". The components of the vector are
- pBluescript (commerical cloning vector plasmid from Stratagene) containing the ampicillin resistance gene and a plasmid origin of replication from Escherichia coli for growth and expression in E. coli
- Mutator1 transposon from Zea mays into which pBluescript was inserted
- a 400 bp marker DNA from Sinorhizobium melioti inserted into RescueMu
No gene is expressed in transgenic maize as a result of this construct. Hint: Avoid abbreviations and spell out everything no matter how mundane.
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