pJTI™ R4 Dest CMV N-EmGFP pA Vector

The pJTI™ R4 Dest CMV N-EmGFP pA vector allows a gene of interest to be expressed as an N-terminal Emerald자세히 알아보기

카탈로그 번호	수량
A14141	100 μg

카탈로그 번호 A14141

제품 가격(KRW)

100 μg

The pJTI™ R4 Dest CMV N-EmGFP pA vector allows a gene of interest to be expressed as an N-terminal Emerald Green Fluorescent Protein (EmGFP) fusion in a Jump-In™ parental cell line. When used in combination with a Jump-In™ parental cell line kit such as the Jump-In™ GripTite™ HEK293 cell line (Cat. No. A14150), an isogeneic stable cell line can be created with less effort and in less time than traditional cell engineering methods. In addition, Gateway™ technology increases the efficiency of the cloning steps to introduce your GOI into the Gateway™-ready pJTI™ R4 Dest CMV N-EmGFP pA vector. The high retargeting efficiency, made possible by the R4 sites in the Jump-In™ parental cell line, allows the use of the isogenic pool for additional experiments without the need for clonal selection. Alternatively, the high retargeting efficiency allows for easy selection of a positive stable clone for expressing your gene of interest with an N-terminal EmGFP.

The Jump-In™ Vectors and Parental Cell Lines Let You:

Quickly and efficiently develop stably engineered isogenic cell pools in about half the time compared to traditional cell engineering methods.
Utilize isogenic expression from a defined genomic locus as the ideal solution for comparative analysis of gene families, isoforms or orthologs.
Generate multiple cell lines in parallel due to the simplified work flow.
Easily access the Jump-In™ technology with the ability to generate an unlimited number of cell lines without complicated licenses or restrictions to interpret.

Save Time with Rapid and Efficient Generation of Engineered Cell Lines

Using Gateway™ technology, you can generate an expression construct for retargeting using the pJTI R4 DEST CMV N-EmGFP pA destination vector. In combination with a Jump-In™ parental cell line kit, the expression cassette is efficiently and specifically inserted into a genomic target R4 site resulting in the generation of functional cell pools in as little as 2 weeks without laborious clone isolation and analysis. Even generation of clonal cell lines can be done with reduced time and effort due to the high percentage of positive clones.

Expand Your Experimental Capabilities

The pJTI™ R4 Dest CMV N-EmGFP pA vector along with a Jump-In™ parental cell line provide the ideal solution for cells and assays where transient engineering technologies are problematic, as well as for difficult to 'engineer' cell lines. The kit also provides a convenient way to create target panels of gene families, isoforms, or orthologs. Genes coding for large proteins or multi-unit proteins are not a problem since the Gateway™ destination vectors accept large inserts.

For Research Use Only. Not for use in diagnostic procedures.

구성 또는 유도성 시스템Constitutive

배달 유형Transfection

용도(애플리케이션)Stable Cell Line Development, Targeted Integration

제품 유형Mammalian Expression Vector

단백질 태그 위치(유전자에 대해)N-terminal

수량100 μg

선택 제제(진핵)None

배송 조건Dry Ice

벡터Jump-In Vectors

클로닝 방법Gateway

농도1.5 μg/μL

제품라인Jump-In

프로모터CMV

단백질 태그GFP (EmGFP)

Unit SizeEach

구성 및 보관

Store vector at -20°C immediately upon receipt.

자주 묻는 질문(FAQ)

When should I consider reversible integration (Flp-In system) vs irreversible integration (Jump-In system)?

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.

What controls do I need in the Jump-In system to check for the successful retargeting of the platform line?

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.

What controls do I need in the Jump-In system to check for the presence of the R4 target site after the creation of the platform cell line?

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.

How much DNA or what controls do I need to include in the Jump-In system in order to get one integration event?

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.

When should I use the Jump-In Fast system vs the Jump-In-TI (Targeted Integration) system?

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.

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