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Protocol for validation of hairpin RNA expression clones

Click on 'figure numbers' or on the words 'details' to read the related description.

 

GST clones validation schema Start Figure 1 Figure 2 Figure 3b Figure 3a Figure 5 Figure 4 Figure 6 Figure 8 Figure 7 Figure 11 Figure 10 Figure 9 Start Figure 1 Figure 2 Figure 3b Figure 3a Figure 5 Figure 6 Figure 11 Figure 8 Figure 7 Figure 10 Figure 9 Figure 4

 

Description

START Bacterial clones
 

The starting material is a collection of pooled E. coli strains carrying the hpRNA expression plasmids, obtained from the partners of the AGRIKOLA project (Belgium, UK, France and Spain) and delivered in multi-well plates.

Figure 1 Plating and growth of bacterial mix
 

To obtain well isolated colonies for each hpRNA expression clone, starting from the original bacterial mixes, one drop of each clone mix is inoculated and spread on a agar plate with solid LB+ medium with kanamycin.

After incubation, the purity of the culture is checked.

Figure 2 Picking and subcultivation of 4 individual bacterial colonies
 

If the purity check is positive, for each hpRNA expression clone 4 independent colonies are picked and transferred in standard 96-well plates.

The viability is checked after incubation.

Figure 3a/b Cell lysis and glycerol stock

 

A fraction of the 4 subcultures is used to make a lysate and simultaneously 75 μl of the subcultures is transferred in 96-well plates filled with 75 μl sterile glycerol to obtain a glycerol stock which will be temporarily stored at −80°C.

Figure 4 PCR-testing

 

As a first step in monitoring the integrity of the artificial GST hairpin gene, a multiplex PCR reaction is done, to confirm that both GST inserts are present and have the expected size.

Because the double LR recombination could produce different intermediates depending on which repeats recombined with each other, the intron spacer in the expression clone either retained its original orientation or was flipped. Because the spacer contained two head-to-head introns, a portion of the hpRNA loop would be spliced out in plant cells regardless of its final orientation, which guarantees efficient silencing.

The four primers (Agri51, Agri56, Agri64, Agri69) were positioned in such a way that the GST subunits present in hpRNA expression plasmids can be easily distinguished by size in DNA agarose gel electrophoresis. Whether the intron spacer was in its original or flipped orientation, the amplified paired DNA fragments were:

  • Agri51-attB1-GST-attB2-Agri56 (primary GST length + 245 bp)
  • Agri64-attB2-GST-attB1-Agri69 (primary GST length + 342 bp)
  • Agri51-attB1-GST-attB2-Agri64 (primary GST length + 442 bp)
  • Agri56-attB2-GST-attB1-Agri69 (primary GST length + 145 bp)

As the plasmids are equally effective with the introns in either orientation, this "intron-flipping" is no more than a cosmetic nuisance.

The fifth larger band generally observed corresponds to the complete hairpin cassette amplified by the outside primers Agri51 and Agri69.

The presence of 5 fragments on the DNA agarose gel indicates the presence of 2 GST inserts.

A GST size within the range 'theoretical GST +/-20%' is considered good.

Only one positive clone out of the four originally picked is selected for further validation.
Figure 5

 Subcultivation of the selected clone from the glycerol stock

 
  • The hpRNA expression clone selected in the previous step is subcultivated on solid LB+ medium with kanamycin, starting from the glycerol stock described in Figure 3b.

  • After incubation, the purity of the culture is checked. The purity check is one of the 3 quality control tests of the BCCM/LMBP quality management system for the deposit of plasmids in the public collection.
Figure 6

 Subcultivation of a single colony from the LB+ agar plate

 
  • One single colony is subcultivated in Greiner tubes, starting from the LB+ agar plate described in Figure 5.

  • After incubation, the viability of the culture is checked. The viability check is one of the 3 quality control tests of the BCCM/LMBP quality management system for the deposit of plasmids in the public collection.

 

Figure 7

reparation and storage of the host/plasmid combination

 

If the viability check is positive, 3 x 0.5 ml of the subculture is transferred in 3 NUNC tubes with 0.5 ml glycerol:

  • 2 NUNC tubes are stored in the BCCM/LMBP stock: 1 in the master stock and 1 in the distribution stock;

  • 1 NUNC tube is stored in the PSB stock.
Figure 8

Preparation of DNA for authenticity test and cryopreservation

 

If the viability check is positive, plasmid DNA is prepared using a robotic platform programmed for this purpose. The protocol is a variation of the alkaline lysis procedure in which the successive steps are performed with an 8-channel liquid handling tool, on a Biomek 2000 Laboratory Automation Workstation, using the Wizard Magnesil Plasmid DNA purification system (Promega).
Figure 9 PCR-testing

 
  • As an extra verification, the plasmid DNA is checked for contamination by a PCR reaction. Simultaneously, the PCR analysis is used to confirm insertion of the attB1-GST-attB2-Pdkintron-Catintron-attB2-GST-attB1 cassette into the hpRNA expression clone which can be indicated by the presence of 5 fragments on the DNA agarose gel.
Figure 10

 Authenticity check by DNA-sequencing + BLAST

 
  • The clean PCR products are sequenced with one BigDye (ABI) reaction and the resulting profile is analyzed with an ABI 3700 capillary automated sequencer.

  • A single sequence reaction and run will be sufficient per clone since the GSTs are 150 to 500 base pairs in length and sequencing runs are routinely informative over 800 bp.

  • Authenticity is checked by blastn homology searches between the experimental DNA sequence derived from the PCR samples and all GSTs sequences in the CATMA database.

  • An alignment length >=60 bp and a DNA sequence homology >95% is considered as a positive confirmation of the GST identity.

  • Small insertion/deletion or point mutations potentially introduced by the successive PCRs are not problematic. The molecules responsible for RNAi are 21 to 23 nt-long small interfering RNAs that are chopped from the larger GST-derived hairpin RNA. Therefore a few point mutations in a GST will not alter its ability to induce silencing as they will not affect the sequence of most siRNAs.

The authenticity check, together with the PCR test, is the third quality control test of the BCCM/LMBP quality management system for the deposit of plasmids in the public collection.
Figure 11 Preparation and storage of DNA

 
  • When the authenticity of the entry plasmid clones can be established, the remaining DNA, prepared in Figure 8, is transferred in 2 multi-well plates and precipitated with ethanol.

  • The two multi-well plates are stored at −20°C on separate locations.
Data input

 
  • To create and complete the database record, a scientific collaborator of BCCM/LMBP checks for each validated GST entry clone whether all necessary data have been provided by PSB to BCCM/LMBP.

  • Creation, completion and finalization of the database record, datasheet and circular map is done by a scientific collaborator of BCCM/LMBP.