TALEN kits The kits are designed to specifically transfer your gene of interest, selection marker or other genetic element into the AAVS1 safe harbor site in human chromosome 19 via TALEN-mediated homologous recombination. The Genome-TALER™ human AAVS1 safe harbor gene knock-in kit includes one pair of AAVS1 TALENs, one donor cloning vector or one donor clone*, one positive control clone and two pairs of knock-in PCR verification primers.
Figure 1. Genome-TALER™ human AAVS1 safe harbor gene knock-in kit. (A) AAVS1 TALENs and donor DNA plasmids
(B) AAVS1 knock-in verification PCR primers
| Cat. No. |
Components |
Description |
Qty |
| TN-AAVS1-L |
AAVS1 left TALEN |
Specifically cleave AAVS1 with target site TN-AAVS1-R to create DSB |
1 |
| TN-AAVS1-R |
AAVS1 right TALEN |
Specifically cleave AAVS1 with target site TN-AAVS1-L ​to create DSB |
1 |
| DC-DON-SH01* |
AAVS1 donor cloning vector |
For cloning GOI or containing GOI
–Two AAVS1 flanking arms for HR
-CMV promoter
-GFP/Puro marker for selection
|
1 |
| DC-RFP-SH01 |
AAVS1 positive control donor clone |
RFP instead of GOI for positive control purpose |
1 |
| HQPAVSHR-5 |
5′ knock-in verification PCR primer pair |
PCR primer pairs for 5’ HR junctions to verify successful integration |
1 |
| HQPAVSHR-3 |
3′ knock-in verification PCR primer pair |
PCR primer pairs for 3’ HR junctions to verify successful integration |
1 |
* DC-DON-SH01 only comes with SH-AVS-K100 kit. Genome-TALER™ AAVS1 ORF knock-in clones can be and ordered separately. Other AAVS1 donor clones can also be custom-made by us.
| (C) |
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| (D) |
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Figure 2. Human genome safe harbor AAVS1 gene targeting (A) AAVS1 RFP control plasmid DC-RFP-SH01 (800 ng) was co-transfected with AAVS1 TALEN pair(600 ng for each) or control TALEN pair into HEK293T cells in a 6-well plate. (B) 48 hr post-transfection, the cells were split 1:10 into a new 6-well plate and selected against 1.0 µg/ml of puromycin. The images were taken after two weeks of selection. Few colonies left in the wells transfected with AAVS1 RFP control + control TALEN. (C) (D) PCR primers designed to amplify the HR junction were used to verify the specific and successful integration. |
Acknowledgement: Design of the AAVS1 left TALEN, AAVS1 right TALEN, and AAVS1 donor control vectors was performed by Dr. Jizhong Zou of the NIH Center for Regenerative Medicine, a Common Fund initiative of the U.S. National Institutes of Health.
CRISPR kits The kits are designed to specifically transfer your gene of interest, selection marker or other genetic element into the AAVS1 safe harbor site in human chromosome 19 via CRISPR-Cas9-mediated homologous recombination. The GeneHero™ human AAVS1 safe harbor gene knock-in kit includes one AAVS1 sgRNA/Cas9 expression clone, one donor cloning vector or one donor clone*, one positive control clone and one knock-in PCR verification kit.
| (A) |
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Figure 3. GeneHero™ human AAVS1 safe harbor gene knock-in kit. (A) AAVS1 sgRNA/Cas9 expression clone and donor DNA plasmids
(B) AAVS1 knock-in verification PCR primers
| Cat. No. |
Components |
Description |
Qty |
| HCP-AAVS1-CG02 |
AAVS1 sgRNA/Cas9 expression clone |
Specifically cleave AAVS1 target site to create DSB |
1 |
| DC-DON-SH01* |
AAVS1 donor cloning vector |
For cloning GOI or containing GOI
–Two AAVS1 flanking arms for HR
-CMV promoter
-GFP/Puro marker for selection
|
1 |
| DC-RFP-SH01 |
AAVS1 positive control donor clone |
RFP instead of GOI for positive control purpose |
1 |
| HQPAVSHR-5 |
5′ knock-in verification PCR primer pair |
PCR primer pairs for 5’ HR junctions to verify successful integration |
1 |
| HQPAVSHR-3 |
3′ knock-in verification PCR primer pair |
PCR primer pairs for 3’ HR junctions to verify successful integration |
1 |
* DC-DON-SH01 only comes with SH-AVS-K200 kit. Genome-TALER™ AAVS1 ORF knock-in clones can be and ordered separately. Other AAVS1 donor clones can also be custom-made by us.
Knock-in clones Human AAVS1 safe harbor knock-in ORF clones More than 20,000 sequence-validated human AAVS1 safe harbor knock-in ORF clones are available. These clones are constructed for specifically integrating the ORF of your interest from a donor plasmid into the AAVS1 safe harbor site in human chromosome 19 by using Genome-TALER™ or GeneHero™ human AAVS1 safe harbor gene knock-in kits.
Figure 4. Map of human AAVS1 safe harbor knock-in ORF clones
Human AAVS1 Safe Harbor knock-in CRISPR-Cas9 clones
Knock-in clone custom services also available
GeneCopoeia also offers AAVS1 donor clone custom services to construct knock-in donor clones containing mutated genes or other genetic elements for targeted AAVS1 integration. To get a quote, please click
here.
Questions? Please contact us at inquiry@genecopoeia.com or 1-301-762-0888.
Figure 5: (A) OCT4 (Cat#: DC-Z0092-SH01) or SOX2(Cat#: DC-T2547-SH01) donor plasmids were co-transfected with AAVS1 TALEN pair into HEK293T cells in a 6-well plate. Cells were sub-cultrured 48 hr post-transefection and selected against puromycin (1µg/ml) for 2 weeks. The expression of CopGFP was checked under a microscope (Nikon Eclipse Ti ) 48h post-transfection or after 2 weeks of drug selection. Few colonies left in cells transfected with donor plasmid alone after puromycin selection (data not shown).
(B) Western blot analysis of proteins from HEK293T cells stably integrated with SOX2(Cat#: DC-T2547-SH01) or OCT4 (Cat#: DC-Z0092-SH01) in AAVS1 site, with cells alone as negative control where endogenous SOX2 or OCT4 protein levels were too low to be detected in the same blot.
Services
AAVS1 donor clone custom services
GeneCopoeia also offers AAVS1 donor clone custom services to construct knock-in donor clones containing mutated genes or other genetic elements for targeted AAVS1 integration. To get a quote, please click
here.
Questions? Please contact us at inquiry@genecopoeia.com or 1-301-762-0888.
AAVS1 stable cell line custom services
| Services |
Description |
Application |
| Stable cell line services |
Monoclonal colony |
Monoclonal stable cell line with TALEN- or CRISPR-Cas9-mediated safe harbor transgene. |
human AAVS1 |
| Cell bank |
Create cell bank of monoclonal stable cell line with TALEN or CRISPR-Cas9-mediated safe harbor transgene. |
human AAVS1 |
Questions? Please contact us at inquiry@genecopoeia.com or 1-301-762-0888.
