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Homologous recombination-mediated cloning and manipulation of genomic DNA regions using Gateway and recombineering systems.

Rozwadowski K, Yang W, Kagale S - BMC Biotechnol. (2008)

Bottom Line: Such expression vectors can be applied to characterise gene regulatory regions through development of reporter-gene fusions, using the Gateway Entry clones of GUS and GFP described here, or for ectopic expression of a coding region cloned into a Gateway Entry vector.We exemplify the utility of this approach with the Arabidopsis PAP85 gene and demonstrate that the expression profile of a PAP85::GUS transgene highly corresponds with native PAP85 expression.Although the system and plasmid vectors described here were developed for applications in plants, the general approach is broadly applicable to gene characterisation studies in many biological systems.

View Article: PubMed Central - HTML - PubMed

Affiliation: Saskatoon Research Centre, Agriculture and Agri-Food Canada, 107 Science Place, Saskatoon, Saskatchewan, Canada, S7N 0X2. RozwadowskiK@agr.gc.ca

ABSTRACT

Background: Employing genomic DNA clones to characterise gene attributes has several advantages over the use of cDNA clones, including the presence of native transcription and translation regulatory sequences as well as a representation of the complete repertoire of potential splice variants encoded by the gene. However, working with genomic DNA clones has traditionally been tedious due to their large size relative to cDNA clones and the presence, absence or position of particular restriction enzyme sites that may complicate conventional in vitro cloning procedures.

Results: To enable efficient cloning and manipulation of genomic DNA fragments for the purposes of gene expression and reporter-gene studies we have combined aspects of the Gateway system and a bacteriophage-based homologous recombination (i.e. recombineering) system. To apply the method for characterising plant genes we developed novel Gateway and plant transformation vectors that are of small size and incorporate selectable markers which enable efficient identification of recombinant clones. We demonstrate that the genomic coding region of a gene can be directly cloned into a Gateway Entry vector by recombineering enabling its subsequent transfer to Gateway Expression vectors. We also demonstrate how the coding and regulatory regions of a gene can be directly cloned into a plant transformation vector by recombineering. This construct was then rapidly converted into a novel Gateway Expression vector incorporating cognate 5' and 3' regulatory regions by using recombineering to replace the intervening coding region with the Gateway Destination cassette. Such expression vectors can be applied to characterise gene regulatory regions through development of reporter-gene fusions, using the Gateway Entry clones of GUS and GFP described here, or for ectopic expression of a coding region cloned into a Gateway Entry vector. We exemplify the utility of this approach with the Arabidopsis PAP85 gene and demonstrate that the expression profile of a PAP85::GUS transgene highly corresponds with native PAP85 expression.

Conclusion: We describe a novel combination of the favourable attributes of the Gateway and recombineering systems to enable efficient cloning and manipulation of genomic DNA clones for more effective characterisation of gene function. Although the system and plasmid vectors described here were developed for applications in plants, the general approach is broadly applicable to gene characterisation studies in many biological systems.

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Rescue of the ABI3 coding region into a Gateway Entry vector. (A) Schematic diagram showing the location of primers and restriction enzyme sites used for analysis of the integrity of candidate clones of pWY148 encoding ABI3 in pJM1. Boxed region represents the ABI3 genomic coding region, and the line represents vector sequence. Arrows indicate location of diagnostic PCR primers and EcoRI sites used for evaluating the correctness of the assembled construct. (B) PCR-based screening of clones potentially encoding ABI3 in pJM1. The 5'-Test (primers Entry-L1 and ABI3-5'-CDS-Rescue-3'-Test) and 3'-Test (primers Entry-L2 and ABI3-3'-Term-Rescue-5'-Test) evaluates for the fusion of the vector with the 5' or 3' end of the ABI3 coding region. An amplicon of 0.4 kb or 0.5 kb is expected for correct vector-gene fusions at the 5' or 3' end, respectively. (C) Restriction enzyme analysis of candidate ABI3 clones in pJM1. Clones 1, 2, 3 and 6 from panel A were digested with EcoRI and exhibit the predicted restriction fragments of 0.8, 2.6 and 3.4 kb. M, DNA size marker (1 kb Plus ladder, Invitrogen) with representation from 0.1–1 kb (B) or 0.65–5 kb (C).
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Figure 2: Rescue of the ABI3 coding region into a Gateway Entry vector. (A) Schematic diagram showing the location of primers and restriction enzyme sites used for analysis of the integrity of candidate clones of pWY148 encoding ABI3 in pJM1. Boxed region represents the ABI3 genomic coding region, and the line represents vector sequence. Arrows indicate location of diagnostic PCR primers and EcoRI sites used for evaluating the correctness of the assembled construct. (B) PCR-based screening of clones potentially encoding ABI3 in pJM1. The 5'-Test (primers Entry-L1 and ABI3-5'-CDS-Rescue-3'-Test) and 3'-Test (primers Entry-L2 and ABI3-3'-Term-Rescue-5'-Test) evaluates for the fusion of the vector with the 5' or 3' end of the ABI3 coding region. An amplicon of 0.4 kb or 0.5 kb is expected for correct vector-gene fusions at the 5' or 3' end, respectively. (C) Restriction enzyme analysis of candidate ABI3 clones in pJM1. Clones 1, 2, 3 and 6 from panel A were digested with EcoRI and exhibit the predicted restriction fragments of 0.8, 2.6 and 3.4 kb. M, DNA size marker (1 kb Plus ladder, Invitrogen) with representation from 0.1–1 kb (B) or 0.65–5 kb (C).

Mentions: To rescue the ABI3 coding region from BAC F22P10, pJM1 was amplified by PCR using the primers ABI3-L1-Rescue-5'CDS and ABI3-L2-Rescue-3'CDS (the sequences of these and all other primers used in this study are listed in Table 1) to incorporate the homology regions required to rescue the ABI3 coding region from BAC F22P10 into the vector. The unique anchor sequences for these primers in pJM1 were chosen to maintain the restriction sites in the multiple cloning site to enable future manipulation of the rescued gDNA sequence by conventional methods, if required. The 5' rescue-homology region encodes 50 bp starting 12 bp upstream of the ABI3 translation start codon, whereas the 3' rescue-homology region encompasses the sequence 329–379 bp 3' of the translation stop codon thereby including the entire 3' UTR and predicted transcription termination signals of ABI3 based on the annotated Arabidopsis genome sequence () [34]. The rescued fragment encoding ABI3 is predicted to be 3262 bp. Despite the size and complexity of the ABI3-rescue primers PCR amplification of the 3.8 kb pJM1 was robust under standard conditions (data not shown). The pJM1 rescue-vector amplicon was then transformed into competent E. coli EL25/F22P10 cells induced to express Red recombinase proteins and candidate clones were selected in the presence of tetracycline. From several hundred tetracycline-resistant colonies sixteen were screened by PCR to assess if the ABI3 gene was rescued into pJM1. The primer pairs were Entry-L1 with ABI3-5'-CDS-Rescue-3'-Test, and Entry-L2 with ABI3-3'-Term-Rescue-5'-Test combining a vector-specific with a gene-specific primer to assess if the 5' and 3' regions, respectively, of ABI3 were present in candidate clones. As shown in Figure 2, PCR tests for 12 of 16 clones resulted in the predicted amplicons of 0.4 kb and 0.5 kb corresponding to the 5' and 3' regions, respectively, of the rescued ABI3 coding region. Four of the rescued ABI3 clones were assessed by restriction enzyme digests using EcoRI and all possessed the predicted fragments of 0.8 and 2.6 kb representing ABI3, and 3.4 kb representing pJM1 (Figure 2). One representative of the four clones was sequenced and shown to encode the sequence of the ABI3 coding region predicted to be subcloned. This clone was designated pWY148.


Homologous recombination-mediated cloning and manipulation of genomic DNA regions using Gateway and recombineering systems.

Rozwadowski K, Yang W, Kagale S - BMC Biotechnol. (2008)

Rescue of the ABI3 coding region into a Gateway Entry vector. (A) Schematic diagram showing the location of primers and restriction enzyme sites used for analysis of the integrity of candidate clones of pWY148 encoding ABI3 in pJM1. Boxed region represents the ABI3 genomic coding region, and the line represents vector sequence. Arrows indicate location of diagnostic PCR primers and EcoRI sites used for evaluating the correctness of the assembled construct. (B) PCR-based screening of clones potentially encoding ABI3 in pJM1. The 5'-Test (primers Entry-L1 and ABI3-5'-CDS-Rescue-3'-Test) and 3'-Test (primers Entry-L2 and ABI3-3'-Term-Rescue-5'-Test) evaluates for the fusion of the vector with the 5' or 3' end of the ABI3 coding region. An amplicon of 0.4 kb or 0.5 kb is expected for correct vector-gene fusions at the 5' or 3' end, respectively. (C) Restriction enzyme analysis of candidate ABI3 clones in pJM1. Clones 1, 2, 3 and 6 from panel A were digested with EcoRI and exhibit the predicted restriction fragments of 0.8, 2.6 and 3.4 kb. M, DNA size marker (1 kb Plus ladder, Invitrogen) with representation from 0.1–1 kb (B) or 0.65–5 kb (C).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2601046&req=5

Figure 2: Rescue of the ABI3 coding region into a Gateway Entry vector. (A) Schematic diagram showing the location of primers and restriction enzyme sites used for analysis of the integrity of candidate clones of pWY148 encoding ABI3 in pJM1. Boxed region represents the ABI3 genomic coding region, and the line represents vector sequence. Arrows indicate location of diagnostic PCR primers and EcoRI sites used for evaluating the correctness of the assembled construct. (B) PCR-based screening of clones potentially encoding ABI3 in pJM1. The 5'-Test (primers Entry-L1 and ABI3-5'-CDS-Rescue-3'-Test) and 3'-Test (primers Entry-L2 and ABI3-3'-Term-Rescue-5'-Test) evaluates for the fusion of the vector with the 5' or 3' end of the ABI3 coding region. An amplicon of 0.4 kb or 0.5 kb is expected for correct vector-gene fusions at the 5' or 3' end, respectively. (C) Restriction enzyme analysis of candidate ABI3 clones in pJM1. Clones 1, 2, 3 and 6 from panel A were digested with EcoRI and exhibit the predicted restriction fragments of 0.8, 2.6 and 3.4 kb. M, DNA size marker (1 kb Plus ladder, Invitrogen) with representation from 0.1–1 kb (B) or 0.65–5 kb (C).
Mentions: To rescue the ABI3 coding region from BAC F22P10, pJM1 was amplified by PCR using the primers ABI3-L1-Rescue-5'CDS and ABI3-L2-Rescue-3'CDS (the sequences of these and all other primers used in this study are listed in Table 1) to incorporate the homology regions required to rescue the ABI3 coding region from BAC F22P10 into the vector. The unique anchor sequences for these primers in pJM1 were chosen to maintain the restriction sites in the multiple cloning site to enable future manipulation of the rescued gDNA sequence by conventional methods, if required. The 5' rescue-homology region encodes 50 bp starting 12 bp upstream of the ABI3 translation start codon, whereas the 3' rescue-homology region encompasses the sequence 329–379 bp 3' of the translation stop codon thereby including the entire 3' UTR and predicted transcription termination signals of ABI3 based on the annotated Arabidopsis genome sequence () [34]. The rescued fragment encoding ABI3 is predicted to be 3262 bp. Despite the size and complexity of the ABI3-rescue primers PCR amplification of the 3.8 kb pJM1 was robust under standard conditions (data not shown). The pJM1 rescue-vector amplicon was then transformed into competent E. coli EL25/F22P10 cells induced to express Red recombinase proteins and candidate clones were selected in the presence of tetracycline. From several hundred tetracycline-resistant colonies sixteen were screened by PCR to assess if the ABI3 gene was rescued into pJM1. The primer pairs were Entry-L1 with ABI3-5'-CDS-Rescue-3'-Test, and Entry-L2 with ABI3-3'-Term-Rescue-5'-Test combining a vector-specific with a gene-specific primer to assess if the 5' and 3' regions, respectively, of ABI3 were present in candidate clones. As shown in Figure 2, PCR tests for 12 of 16 clones resulted in the predicted amplicons of 0.4 kb and 0.5 kb corresponding to the 5' and 3' regions, respectively, of the rescued ABI3 coding region. Four of the rescued ABI3 clones were assessed by restriction enzyme digests using EcoRI and all possessed the predicted fragments of 0.8 and 2.6 kb representing ABI3, and 3.4 kb representing pJM1 (Figure 2). One representative of the four clones was sequenced and shown to encode the sequence of the ABI3 coding region predicted to be subcloned. This clone was designated pWY148.

Bottom Line: Such expression vectors can be applied to characterise gene regulatory regions through development of reporter-gene fusions, using the Gateway Entry clones of GUS and GFP described here, or for ectopic expression of a coding region cloned into a Gateway Entry vector.We exemplify the utility of this approach with the Arabidopsis PAP85 gene and demonstrate that the expression profile of a PAP85::GUS transgene highly corresponds with native PAP85 expression.Although the system and plasmid vectors described here were developed for applications in plants, the general approach is broadly applicable to gene characterisation studies in many biological systems.

View Article: PubMed Central - HTML - PubMed

Affiliation: Saskatoon Research Centre, Agriculture and Agri-Food Canada, 107 Science Place, Saskatoon, Saskatchewan, Canada, S7N 0X2. RozwadowskiK@agr.gc.ca

ABSTRACT

Background: Employing genomic DNA clones to characterise gene attributes has several advantages over the use of cDNA clones, including the presence of native transcription and translation regulatory sequences as well as a representation of the complete repertoire of potential splice variants encoded by the gene. However, working with genomic DNA clones has traditionally been tedious due to their large size relative to cDNA clones and the presence, absence or position of particular restriction enzyme sites that may complicate conventional in vitro cloning procedures.

Results: To enable efficient cloning and manipulation of genomic DNA fragments for the purposes of gene expression and reporter-gene studies we have combined aspects of the Gateway system and a bacteriophage-based homologous recombination (i.e. recombineering) system. To apply the method for characterising plant genes we developed novel Gateway and plant transformation vectors that are of small size and incorporate selectable markers which enable efficient identification of recombinant clones. We demonstrate that the genomic coding region of a gene can be directly cloned into a Gateway Entry vector by recombineering enabling its subsequent transfer to Gateway Expression vectors. We also demonstrate how the coding and regulatory regions of a gene can be directly cloned into a plant transformation vector by recombineering. This construct was then rapidly converted into a novel Gateway Expression vector incorporating cognate 5' and 3' regulatory regions by using recombineering to replace the intervening coding region with the Gateway Destination cassette. Such expression vectors can be applied to characterise gene regulatory regions through development of reporter-gene fusions, using the Gateway Entry clones of GUS and GFP described here, or for ectopic expression of a coding region cloned into a Gateway Entry vector. We exemplify the utility of this approach with the Arabidopsis PAP85 gene and demonstrate that the expression profile of a PAP85::GUS transgene highly corresponds with native PAP85 expression.

Conclusion: We describe a novel combination of the favourable attributes of the Gateway and recombineering systems to enable efficient cloning and manipulation of genomic DNA clones for more effective characterisation of gene function. Although the system and plasmid vectors described here were developed for applications in plants, the general approach is broadly applicable to gene characterisation studies in many biological systems.

Show MeSH
Related in: MedlinePlus