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View additional product information for Jump-In™ Fast Gateway™ System - FAQs (A10893)
21 product FAQs found
In the single-step protocol for the BP/LR Clonase reaction, we would not recommend substituting the BP Clonase II/LR Clonase II enzymes with BP Clonase /LR Clonase enzymes as this would result in very low recombination efficiency.
Yes, we have come up with a single-step protocol for BP/LR Clonase reaction (http://www.thermofisher.com/us/en/home/life-science/cloning/gateway-cloning.html#1), where DNA fragments can be cloned into Destination vectors in a single step reaction, allowing you to save time and money.
We would recommend performing a BP reaction with a Donor vector in order to obtain an entry clone. This entry clone can then be used in an LR reaction with the Destination vector to obtain the new expression clone.
We do not offer the 5X LR Clonase buffer and 5X BP Clonase buffer as standalone products. They are available as part of the enzyme kits.
We do not offer any Gateway vectors for expression in plants.
We would recommend engineering an expression marker/reporter in the plasmid used to create the platform line, and then screening the platform line for expression of this marker to identify a high-expressing locus. Otherwise, the process can get quite labor-intensive, as multiple lines would have to be screened after retargeting.
The pJTI Phic31 Int vector does not contain an NLS. Adding an NLS could increase the efficiency of site-specific integration at pseudo attP sites, but there are no data to support it. There is one paper describing the use of an NLS on a PhiC31 integrase vector, but the authors didn't measure integration into pseudo attP sites.
The amount of DNA to be used to obtain single copies should be determined by control experiments done in the absence of integrase. The same amount of DNA that yields less than 5 colonies in the absence of integrase should be used in the presence of integrase. Typically, the integrase expression plasmid makes up most of the amount of DNA used for transfection.
Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.
We recommend using the Jump-In Fast system if you need stable mammalian expression and want to quickly generate well-expressing clones. You can have well-expressing clones with one or more integrations at the PhiC31 pseudo-att P sites. A Southern blot is necessary to confirm the number of integrated events. Use the Jump-InTI system if you need isogenic expression, where every cloned gene would be expressed from the same locus in the same background, with no chromosomal position effects.
The Jump-In system is PhiC31-integrase mediated and is a stable, targeted, and irreversible mammalian expression system. It consists of the Jump-In Fast system that involves a single integration step and the Jump-InTI (targeted integration) system that needs two integration steps, both of which are targeted and irreversible. In contrast, the Flp-In system is a stable, targeted mammalian expression system that is reversible. The first integration is random (integration of pFRT/lacZeo), and the second integration (integration of the Flp-In expression vector) is targeted but reversible.
We recommend using One Shot ccdB Survival 2 T1R Competent Cells, Cat. No. A10460. This strain is resistant to the toxic effects of the ccdB gene. Note: Do not use general E. coli cloning strains, including TOP10 or DH5alpha, for propagation and maintenance, as these strains are sensitive to ccdB effects.
The consensus Kozak sequence is A/G NNATGG, where the ATG indicates the initiation codon. Point mutations in the nucleotides surrounding the ATG have been shown to modulate translation efficiency. Although we make a general recommendation to include a Kozak consensus sequence, the necessity depends on the gene of interest and often, the ATG alone may be sufficient for efficient translation initiation. The best advice is to keep the native start site found in the cDNA unless one knows that it is not functionally ideal. If concerned about expression, it is advisable to test two constructs, one with the native start site and the other with a consensus Kozak. In general, all expression vectors that have an N-terminal fusion will already have an initiation site for translation.
Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.
ATG is often sufficient for efficient translation initiation although it depends upon the gene of interest. The best advice is to keep the native start site found in the cDNA unless one knows that it is not functionally ideal. If concerned about expression, it is advisable to test two constructs, one with the native start site and the other with a Shine Dalgarno sequence/RBS or consensus Kozak sequence (ACCAUGG), as the case may be. In general, all expression vectors that have an N-terminal fusion will already have a RBS or initiation site for translation.
Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.
Use irreversible integration (Jump-In system) if the transgene should be sustained in the mammalian genome for a long time. Use reversible integration such as Flp-In system if the transgene needs to be replaced with another gene of interest after a short period of time.
The second step in targeted integration is the retargeting event mediated by the R4 integrase where the genetic elements of interest are site-specifically transferred from the retargeting expression construct (created using the MultiSite Gateway Pro module) onto the genome of the platform line. This integration event also positions the EF1alpha promoter upstream of the blasticidin, neomycin, or eosin resistance gene (i.e., "promoterless" selection marker), thus allowing the selection of transformants that are successfully "retargeted" using the appropriate selection agent. Although you select from successfully retargeted clones using the blasticidin, Geneticin, or Zeocin antibiotic, you may also perform a nested PCR to amplify the region from the EF1alpha promoter to the appropriate resistance gene. You can amplify the hygromycin resistance gene as a positive control. Similar to the platform line creation, you may also perform a Southern blot analysis with a probe designed for your gene of interest.
A platform cell line is created when the R4 attP retargeting sequences are site-specifically inserted into the mammalian genome via PhiC31 Int-mediated recombination. In addition to the R4 retargeting sequences, this integration event introduces the hygromycin resistance gene under the control of the HSV TK promoter and the promoterless Bsd, Neo, or Zeo resistance marker, depending on the platform vector used (i.e., pJTI/Bsd, pJTI/Neo, or pJTI/Zeo). Although you select for transformants carrying the R4 retargeting sequences by their resistance to hygromycin, you may perform PCR analysis to check the integrity of the R4 attP retargeting sequences. For this, we recommend amplifying the region from the R4 attP sequence to the appropriate resistance marker (depending on the platform line used) using the genomic DNA from the platform line. A nested PCR is recommended to reduce the high background you may observe with only primary PCR. Alternatively, you may create a labeled DNA probe by PCR amplifying an approximately 1.5 kb region covering the retargeting sequences, and then perform a Southern blot analysis. The Southern blot will also act as an additional check to verify that only a single copy of the retargeting sequence is integrated into the genome.
The amount of DNA to be used to obtain single copies should be determined by control experiments done in the absence of integrase. The same amount of DNA that yields less than 5 colonies in the absence of integrase should be used in the presence of integrase. Typically, the integrase expression plasmid makes up most of the amount of DNA used for transfection.
Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.
Use the Jump-In Fast system if you need stable mammalian expression and want to quickly generate well-expressing clones. You can have well-expressing clones with one or more integrations at the PhiC31 pseudo-att P sites. A Southern blot is necessary to confirm the number of integrated events.
Use the Jump-In TI system if you need isogenic expression, where every cloned gene would be expressed from the same locus in the same background with no chromosomal position effects.
The Jump-In system is PhiC31-integrase mediated and is a stable, targeted, and irreversible mammalian expression system, involving one integration step. The Jump-In TI (Targeted Integration) system needs two integration steps, both of which are targeted and irreversible. In contrast, the Flp-In system is a stable, targeted mammalian expression system that is reversible. The first integration is random (integration of pFRT/lacZeo) and the second integration (integration of the Flp-In expression vector) is targeted but reversible.
Prokaryotic mRNAs contain a Shine-Dalgarno sequence, also known as a ribosome binding site (RBS), which is composed of the polypurine sequence AGGAGG located just 5’ of the AUG initiation codon. This sequence allows the message to bind efficiently to the ribosome due to its complementarity with the 3’-end of the 16S rRNA. Similarly, eukaryotic (and specifically mammalian) mRNA also contains sequence information important for efficient translation. However, this sequence, termed a Kozak sequence, is not a true ribosome binding site, but rather a translation initiation enhancer. The Kozak consensus sequence is ACCAUGG, where AUG is the initiation codon. A purine (A/G) in position -3 has a dominant effect; with a pyrimidine (C/T) in position -3, translation becomes more sensitive to changes in positions -1, -2, and +4. Expression levels can be reduced up to 95% when the -3 position is changed from a purine to pyrimidine. The +4 position has less influence on expression levels where approximately 50% reduction is seen. See the following references:
- Kozak, M. (1986) Point mutations define a sequence flanking the AUG initiator codon that modulates translation by eukaryotic ribosomes. Cell 44, 283-292.
- Kozak, M. (1987) At least six nucleotides preceding the AUG initiator codon enhance translation in mammalian cells. J. Mol. Biol. 196, 947-950.
- Kozak, M. (1987) An analysis of 5´-noncoding sequences from 699 vertebrate messenger RNAs. Nucleic Acids Res. 15, 8125-8148.
- Kozak, M. (1989) The scanning model for translation: An update. J. Cell Biol. 108, 229-241.
- Kozak, M. (1990) Evaluation of the fidelity of initiation of translation in reticulocyte lysates from commercial sources. Nucleic Acids Res. 18, 2828.
Note: The optimal Kozak sequence for Drosophila differs slightly, and yeast do not follow this rule at all. See the following references:
- Romanos, M.A., Scorer, C.A., Clare, J.J. (1992) Foreign gene expression in yeast: a review. Yeast 8, 423-488.
- Cavaneer, D.R. (1987) Comparison of the consensus sequence flanking translational start sites in Drosophila and vertebrates. Nucleic Acids Res. 15, 1353-1361.
Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.
Eukaryotic (and specifically mammalian) mRNA contains sequence information that is important for efficient translation. However, this sequence, termed a Kozak sequence, is not a true ribosome binding site, but rather a translation initiation enhancer. The Kozak consensus sequence is ACCAUGG, where AUG is the initiation codon. A purine (A/G) in position -3 has a dominant effect; with a pyrimidine (C/T) in position -3, translation becomes more sensitive to changes in positions -1, -2, and +4. Expression levels can be reduced up to 95% when the -3 position is changed from a purine to pyrimidine. The +4 position has less influence on expression levels where approximately 50% reduction is seen. See the following references:
Kozak, M. (1986) Point mutations define a sequence flanking the AUG initiator codon that modulates translation by eukaryotic ribosomes. Cell 44, 283-292.
Kozak, M. (1987) At least six nucleotides preceding the AUG initiator codon enhance translation in mammalian cells. J. Mol. Biol. 196, 947-950.
Kozak, M. (1987) An analysis of 5´-noncoding sequences from 699 vertebrate messenger RNAs. Nucleic Acids Res. 15, 8125-8148.
Kozak, M. (1989) The scanning model for translation: An update. J. Cell Biol. 108, 229-241.
Kozak, M. (1990) Evaluation of the fidelity of initiation of translation in reticulocyte lysates from commercial sources. Nucleic Acids Res. 18, 2828.
Note: The optimal Kozak sequence for Drosophila differs slightly, and yeast do not follow this rule at all. See the following references:
Romanos, M.A., Scorer, C.A., Clare, J.J. (1992) Foreign gene expression in yeast: a review. Yeast 8, 423-488.
Cavaneer, D.R. (1987) Comparison of the consensus sequence flanking translational start sites in Drosophila and vertebrates. Nucleic Acids Res. 15, 1353-1361.
Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.