The identification of differentially expressed genes to investigate a specific developmental system or the genetic changes associated with a given pathology is one of the most commonly pursued tasks in molecular and cellular biology. Diverse techniques allow the study of the modulation of gene expression including differential display (DD)-PCR. DD-PCR permits the isolation of cDNA reverse transcribed from mRNA, allowing comparison of gene expression of differentially treated samples. The cDNA fragments are cloned and identified by sequencing and homology searches. The differential expression of the cDNA can then be confirmed using more stringent techniques for the evaluation of steady-state mRNA such as Northern blots and semi-quantitative PCR.
We are currently analyzing gene expression in cultured breast cancer cells in response to microenvironmental modulators such as parathyroid hormone-related peptide (PTHrP), which is known to affect cell proliferation and invasivity(1)
. Using mRNA DD-electrophoretograms and re-amplifying and purifying selected cDNA fragments(3)
, we have obtained a number of DNA preparations suitable for sequencing and subsequent identification of the parental transcript.
Here we report the successful use of LigaFast™ Rapid DNA Ligation System and pGEM®-T Easy Vector System (Cat.# A1360) for cloning PCR products. This procedure yields enough recombinant plasmid for sequence analysis.
Results and Discussion
To identify genes selectively regulated in a breast cancer cell line after treatment with a midregion fragment of PTHrP, we isolated cDNA fragments by DD-PCR(3)
. The cDNA fragments were ligated into pGEM®-T Easy Vector. When working with DD-PCR insert-containing vectors, we found that plating 300–500µl of transformed bacterial cells generated a higher number of colonies with a consequent higher percentage of white colonies. Additionally, the 6:1 insert:plasmid ratio yielded a 50% increase of colony number. On the other hand, when the control DNA fragment was used for transformation, an insert:plasmid ratio of 3:1 and 100µl of transformation culture gave optimal results, (positive control yielded more than 90% white colonies). The number of colonies grown in the background control was negligible.
Figure 1 shows the electrophoretic analysis of recombinant plasmids purified from the cell lysates; we were able to obtain 1-5µg of recombinant plasmid from 5ml cultures, an amount suitable for automated sequencing. Figure 2 shows the restriction analysis of recombinant plasmids digested with EcoR I, whose recognition site closely flanks the insertion site. We found that after enzymatic treatment, DNA fragments corresponded in size to those of the DD-PCR bands plus the remnant region of the polylinker.
We compared the colony-forming efficiency of three different bacterial strains, JM109, HB101 and XL1 Blue. JM109 cells yielded 20% more colonies, both blue and white, than the other two cell types. We have also successfully cloned DNA fragments extracted and purified from agarose gel bands.
In conclusion, we can recommend the pGEM®-T Easy Vector System, in conjunction with LigaFast™ Rapid DNA Ligation System, as a versatile tool for the quick cloning of DD-PCR products and the production of recombinant plasmids for subsequent sequencing.
Materials and Methods
Preparation of cDNA fragments: We amplified cDNA fragments (180-750bp) obtained from DD-PCR using PCR Master Mix (Cat.# M7501). Annealing temperatures and cycling profiles were empirically determined for each reaction. For the A-tailing reaction, the elongation time of the last cycle was prolonged to 20 minutes. We determined the purity and the amount of the PCR products by agarose gel electrophoresis.
Ligation and transformation reactions: The ligation reaction was set up using pGEM®-T Easy vector and LigaFast™ Rapid DNA Ligation System using a PCR product:vector molar ratio of 3:1 or 6:1. Positive and background controls were prepared in parallel as recommended by the manufacturer(4)
. The reactions were incubated overnight at 4°C, after which they were quickly centrifuged and 2µl added to 50µl of either JM109, HB101 (Cat.# L2011) or XL1 Blue (kind gift of Prof. Ida Albanese and Mario La Farina) competent cells. Transformation was performed according to manufacturer’s instructions(4)
onto LB/Ampicillin/IPTG/X-Gal plates.
Plasmid extraction: White bacterial colonies were picked and grown overnight at 37°C with agitation in 5ml Luria broth supplemented with 25µl Ampicillin (100µg/ml). Polypropylene tubes were used for colony incubation. Recombinant plasmids were purified using a commercial miniprep kit and quantitated by gel electrophoresis. Insertion of the PCR product of interest into the plasmid was verified by EcoR I digestion of 200–400ng of recombinant plasmid and analysis by agarose gel electrophoresis.
Work partially supported by Italian MURST (Cofin and ex 60%).