MOGEN promoter-trapping lines donated to NASC
P.C. Sijmons
MOGEN int NV,
Einsteinweg 97,
2333 CB Leiden,
The Netherlands
phone +31-71-258282
fax +31-71-221471
e-mail dekker_b@rulgca.leidenuniv.nl
In September 1994, almost 800 transgenic Arabidopsis lines from MOGEN (Leiden, The Netherlands) were deposited at the Nottingham Arabidopsis Stock Centre. The lines carry at least one T-DNA insert from pMOG553 which has a promoterless GUS gene close to the right border sequence and a hygromycin resistance marker. The lines can be used for research purposes and will be available initially from the Nottingham Arabidopsis Stock Centre after signing a specific release form. The origin of the pMOG533/Arabidopsis lines must be duly acknowledged when used in publications.
These lines will be released by NASC as soon as possible
To facilitate molecular analysis of the MOGEN lines by other research groups, I have included all relevant data that is presently available (construct details, transformation protocol used, etc.)
Origin
The deposited lines come from a collection of over 1250 independent transformants that were originally made to study gene regulation in nematode-feeding structures. Its use is first described in:
Goddijn, O.J.M. Lindsey, K. Vanderlee, F.M. Klap, J.C. & Sijmons, P.C.
Differential gene expression in nematode-induced feeding structures of transgenic plants harbouring promoter gusA fusion constructs. Plant J. (1993) 4:863-873.
Transformation
The Arabidopsis ecotype C24 (Lehle Seeds, Arizona, USA) was used for the root transformation protocol described by Valvekens et al (PNAS, 85:5536-5540, 1988) with minor alterations. Seeds were germinated in liquid germination medium (appendix 1) for 1 week (100rpm rotary shaker, 23C, 1000 lux (16h L / 8h D). Roots harvested from the germinated seedlings were directly used for co cultivation with Agrobacterium tumefaciens (strain MOG101, C58 chromosomal background, binary vector pMOG553). Hygromycin shoots were allowed to root and transferred to soil.
Plant Growth & Seed Production
All plant growth was done in growth chambers (16h L / 8h D) at 21C and ca. 40-60% RH in Aracon tubes (Beta Developments, Gent, Belgium, phone +32-9-251-7375) to prevent cross-pollination. The primary transformants were grown to maturity and harvested individually (S1 seeds). Seeds were stored at 10% RH, 8C until further analysis (see below). For bulking, seeds were plated without surface sterilization on a selective germination medium (GM + 10mg/ml hygromycin) and kept at 4C for 4 days and were then transferred to RT with moderate light conditions for ca. 10 days. Usually 8-10 hygromycin resistant seedlings were transferred to potting soil and grown to maturity in Aracon tubes. The seeds harvested from the 8-10 plants (S2 seeds) were pooled. It is from these S2 batches that aliquots were deposited in Nottingham. The aliquots thus contain both heterozygote and homozygote seeds.
GUS analysis and available data
Most of the presently available data was recorded through analysis of seedlings from S1 seeds, except for those lines that yielded only very few S1 seeds; these were bulked prior to GUS analysis. When released all the information generated through the analysis of these lines by MOGEN will be made public. This information includes:
- segregation of S1 seeds on hygromycin
- GUS expression at seedling stage (analyzed just prior to soil transfer of the S1 seeds), roughly divided into tissue classes.
- GUS analysis was performed using the standard Jefferson histochemical X-Glu assay at 37¡C, 1-16 h. The tissues were cleared with 70% EtOH.
- maximum GUS expression (on a scale from + to +++++). This indicates the maximum level observed in the plant in any one tissue. Thus a line can be GUS positive in the root vascular cylinder as well as the primary leaves; both tissues will be marked but only the highest expression is indicated on the scale. Additional information will also be supplied in the remarks section.
- a limited number of plants have been analyzed during growth in soil up until flowering.
- for a limited number of lines the number of T-DNA inserts have been determined with Southern analysis (see notes).
Non-available lines
A number of lines are not available through Nottingham. These lines were either hygromycin-sensitive escapes or identified in a screen by us or by our collaborators and are used for more detailed research. I expect that they will become available after publication or when we are notified that they can be released into the public domain. The following categories (or selections thereof) are not yet available:
- nematode-inducible GUS expression
- roottip GUS expression (meristem, cap-cells, lateral root intitiation etc.)
- constitutive GUS expression
- lines that are sterile when homozygous
- hormone-inducible GUS expression
- lines that have not yet been bulked to S2 (a second batch of ca. 250 lines will be send in 1995)
Important Notes
- Although plant growth and seed harvest have been done with the utmost care (and with much help from the Aracon tubes), handling such high numbers of individual lines over a period of 2 years by several people is not without risk. Therefore, we can not guarentee that there is no cross contamination at all between different lines. For example during HygR analysis of S1 seeds, sometimes very low numbers of HygR seedlings were counted. We do not know at this stage whether this is the result of a chimaeric primary transformant or that it is contamination from another batch.
- during the tissue culture phase, in general only one shoot per callus was harvested. Again because of the number of people involved in the project over the years, there may be individual lines in the collection that originally stem from the same transformed cell.
- hygromycin resistance was scored ca. 10-14 days after germination. Since the seeds were sprinkled manually across the agar plates and sometimes were in clusters, but possibly also due to position effects of the integrated T-DNA, lines had different degrees of hygromycin resistance. On top of that HygR was scored by different individuals. Therefore the indicated numbers for segregation should only be used as an indication (especially for the early lines with a low number).
- 3:1 segregation ratio only indicates a single locus, not necessarily a single T-DNA. Southern analysis of 30 different 3:1 segregating lines showed that only 9 had a single insert. Such lines are optimal for invertedPCR isolation of the tag. However, when a plant contains more than one insert and the expression pattern is interesting enough, all T-DNA tags can be isolated if necessary. Our experiance is that it is usually fastest to make a genomic library for that particular line. It is difficult to use iPCR on more than two tags.
- we have observed differences between GUS expression at seedling stage and after transfer to soil; one should be careful to compare GUS data between different growth conditions or developmental stages.
Please send research data that is of general interest (segregation analysis, number of T-DNA inserts etc.) as much as possible to the Nottingham Stock Centre. It will be used to keep the database up-to-date.
In case you want some more detailed information, you can contact:
Dr. Stephan Ohl Dr. Peter C. Sijmons
MOGEN international NV Current Address: ATO-DLO
Einsteinweg 97 P.O.box 17
2333 CB LEIDEN 6700 AA Wageningen
The Netherlands The Netherlands
fax +31-71-221471 fax +31-8370-12260
dekker_b@rulgca.leidenuniv.nl p.c.sijmons@ato.agro.nl