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Mouse ROSA26 safe harbor gene knock-in kits and clones

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Safe Harbor Kits

Introduction

ROSA26 (also known as ROSAβgeo26 locus) in the mouse genome is first found in chromosome 6 in one particular strain of mice-named ROSAβgeo26-expressed β galactosidase from a randomly inserted transgene at high levels uniformly in nearly all tissues examined. This locus expresses one coding transcript and two noncoding transcripts, and only the non-coding transcripts are disrupted by the insertion. While pups homozygous for the insertion are born at slightly lower frequency than heterozygous pups, homozygotes appeared to develop normally and were fertile. Thererore, the “Rosa26” locus has since been used as a transgene insertion site that causes no apparent adverse effects on fitness, and permits stable gene expression.

ROSA26-specific TALENs or CRISPR-Cas9 can generate a DNA double-strand break (DSB) at ROSA26 in the mouse genome, stimulating natural DNA repair mechanisms. In the presence of ROSA26 ORF knockin clones, homologous recombination (HR) occurs and integrates DNA fragments from knockin clones into the safe harbor locus.

 
 
 
 

 

 

 

 

 

Mouse ROSA26 safe harbor knockin kits

Buy Catalog # Product Description Price
SH-ROS-K100 Genome-TALER™
mouse ROSA26 safe harbor gene knock-in kit
Includes:
ROSA26 TALEN pair (TN-ROSA26)
ROSA26 donor cloning vector (DC-DON-SH02)
ROSA26 positive control donor (DC-RFP-SH02)
knock-in verification primer pairs (MQPROSHR)
$1895
SH-ROS-K000 Genome-TALER™
mouse ROSA26 safe harbor gene knock-in kit
(without donor)
Includes:
ROSA26 TALEN pair (TN-ROSA26)
ROSA26 positive control (DC-RFP-SH02)
knock-in verification primer pairs (MQPROSHR)
$1695
SH-ROS-K200 Genome-CRISP™
mouse ROSA26 safe harbor gene knock-in kit
Includes:
ROSA26 sgRNA/Cas9 expression clone (MCP-ROSA26-CG01)
ROSA26 donor cloning vector (DC-DON-SH02)
ROSA26 positive control donor (DC-RFP-SH02)
knock-in verification primer pairs (MQPROSHR)
$1895
SH-ROS-K002 Genome-CRISP™
mouse ROSA26 safe harbor gene knock-in kit
(without donor)
Includes:
ROSA26 sgRNA/Cas9 expression clone (MCP-ROSA26-CG01)
ROSA26 positive control (DC-RFP-SH02)
knock-in verification primer pairs (MQPROSHR)
$1695

 

Mouse ROSA26 Safe Harbor knockin ORF clones

Catalog # Product Description Price
Variable Mouse ROSA26 knockin ORF donor clones Mouse ROSA26 knock-in ORF donor clone containing CMV-driven ORF of customer's gene of interest

 

Mouse ROSA26 Safe Harbor knockin CRISPR-Cas9 clones

Important Note: The vector-specifc knock-in verification primers provided with the Mouse ROSA26 Safe Harbor knockin kits (catalog numbers SH-ROS-K100, SH-ROS-K000, SH-ROS-K200, and SH-ROS-K002) are not compatible with the Mouse ROSA26 Safe Harbor knockin CRISPR-Cas9 clones in the table below. If you purchase these clones with our Mouse Safe Harbor knockin kits, you will be provided with clone-specific knockin verification primers.

 

Buy Catalog # Product Application Selection marker Promoter Cas9 integration site Delivery format Price
DC-C9NU-06 Cas9 ROSA26 knockin donor clone To knockin Cas9 nuclease gene to mouse ROSA26 safe harbor site using CRISPR or TALEN Puro CBh ROSA26 Bacterial stock or transfection-ready DNA $495
DC-C9NU-07 Cas9 ROSA26 knockin donor clone Hygro CBh ROSA26 $495
DC-C9NU-08 Cas9 ROSA26 knockin donor clone Neo CBh ROSA26 $495
DC-C9NU-12 Inducible Cas9 AAVS1 knockin donor clone Puro TRE3G ROSA26 $495

 

TALEN Kits

The kits are designed to specifically transfer your gene of interest, selection marker or other genetic element into the ROSA26 safe harbor site in mouse genome via TALEN-mediated homologous recombination.

The Genome-TALER™ mouse ROSA26 safe harbor gene knock-in kit includes one pair of ROSA26 TALENs, one donor cloning vector or one donor clone*, one positive control clone and one knock-in PCR verification kit.

(A)  
mROSA26-left-talen mROSA26-right-talen
     
mROSA26-donor-control pDonor-ORF-sh02  
(B)

 

Figure 2. Genome-TALER™ mouse ROSA26 safe harbor gene knock-in kit. (A) ROSA26 TALEN and donor DNA plasmids (B)ROSA26 knock-in verification PCR primers

 

Cat. No.  Components Description Qty

 TN-ROSA26-L 

ROSA26 left TALEN

Specifically cleave ROSA26 with target site TN-ROSA26-R to create DSB 1

 TN-ROSA26-R 

ROSA26 right TALEN

Specifically cleave ROSA26 with target site TN-ROSA26-L to create DSB 1

 DC-DON-SH02* 

ROSA26 donor cloning vector

For cloning GOI or containing GOI  

-Two ROSA26 flanking arms for HR
-CMV promoter
-GFP/Puro marker for selection

1

 DC-RFP-SH02

ROSA26 positive control donor clone RFP instead of GOI for positive control purpose 1

 MQPROSHR-5

5' knock-in verification PCR primer pair PCR primer pairs for 5’ HR junctions to verify successful integration 1

 MQPROSHR-3

3' knock-in verification PCR primer pair PCR primer pairs for 3’ HR junctions to verify successful integration 1

* DC-DON-SH02 only comes with SH-ROS-K100 kit. ROSA26 ORF knock-in clones and other ROSA26 donor clones can be custom-made by us.

 

(A) (B)
The_comparison_chart1-s
(C)
 

Figure 3. Mouse genome safe harbor ROSA26 gene targeting

(A) ROSA26 RFP control plasmid DC-RFP-SH02 (800 ng) was co-transfected with ROSA26 TALEN pair (600 ng for each) or control only DC-RFP-SH02 (800 ng) transfected into mouse Neuro2a 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 only ROSA26 RFP control.

(C) PCR primers designed to amplify the HR junctions were used to verify the specific and successful integration.

CRISPR kits

The kits are designed to specifically transfer your gene of interest, selection marker or other genetic element into the ROSA26 safe harbor site in mouse genome via CRISPR-Cas9-mediated homologous recombination.

The Genome-CRISP™ mouse ROSA26 safe harbor gene knock-in kit includes a ROSA26 sgRNA/Cas9 expression clone, one donor cloning vector or one donor clone*, one positive control clone and one knock-in PCR verification kit.

(A)  
mROSA26-right-talen
     
mROSA26-donor-control pDonor-ORF-sh02  
(B)

 

Figure 4. The Genome-CRISP™ mouse ROSA26 Safe harbor gene knock-in kit. (A) ROSA26 sgRNA/Cas9 expression clone and donor DNA plasmids (B)Knock-in verification PCR primers

 

Cat. No.  Components Description Qty

 MCP-ROSA26-CG01 

ROSA26 sgRNA/Cas9 expression clone

Specifically cleave ROSA26 target site to create DSB 1

 DC-DON-SH02* 

ROSA26 donor cloning vector

For cloning GOI or containing GOI  

-Two ROSA26 flanking arms for HR
-CMV promoter
-GFP/Puro marker for selection

1

 DC-RFP-SH02

ROSA26 positive control donor clone RFP instead of GOI for positive control purpose 1

 MQPROSHR-5

5' knock-in verification PCR primer pair PCR primer pairs for 5’ HR junctions to verify successful integration 1

 MQPROSHR-3

3' knock-in verification PCR primer pair PCR primer pairs for 3’ HR junctions to verify successful integration 1

* DC-DON-SH02 only comes with SH-ROS-K200 kits. ROSA26 ORF knock-in clones and other ROSA26 donor clones can be custom-made by us.

 

(A) (B)
The_comparison_chart1-s
(C)
 

Figure 5. Mouse genome safe harbor ROSA26 gene targeting

(A)ROSA26 RFP control plasmid DC-RFP-SH02 (800 ng) was co-transfected with ROSA26 sgRNA/Cas9 expression clone (600 ng) or control of only control plasmid DC-RFP-SH02 (800 ng) transfected into mouse Neuro2a 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 only ROSA26 RFP control.

(C) PCR primers designed to amplify the HR junctions were used to verify the specific and successful integration.

Services

mROSA26 donor clone custom services

GeneCopoeia also offers mROSA26 donor clone custom services to construct knock-in donor clones containing mutated genes or other genetic elements for targeted mROSA26 integration. To get a quote, please click here.

Questions? Please contact us at inquiry@genecopoeia.com or 1-866-360-9531.

 

Services Description Application
Stable cell line services Monoclonal colony Monoclonal stable cell line with TALEN- or CRISPR-Cas9-mediated safe harbor transgene. mouse ROSA26 
Cell bank Create cell bank of monoclonal stable cell line with TALEN- or CRISPR-Cas9-mediated safe harbor transgene. mouse ROSA26 

Transgenic mouse services

Transgenic mouse

Transgenic mice with TALEN- or CRISPR-Cas9-mediated ROSA26 transgene.

mouse ROSA26 

Related Products

Human AAVS1 safe harbor knockin kits and clones

Genome-TALER™ or Genome-CRISP™ human AAVS1 safe harbor gene knock-in kit – TALEN or CRISPR-Cas9-mediated gene knock-in kits designed to specifically transfer your gene of interest, selection marker or other genetic element into the AAVS1 safe harbor site in human chromosome 19. More than 20,000 AAVS1 compatible ORF donor clones are also available. The safe integration ensures transcription-competency of the transgenes and presents no known adverse effect.

 

TALE and CRISPR-Cas9 products and services

Genome-TALER™ TALEN and TALE-TF custom services – GeneCopoeia offers several levels of affordable custom services for designing, creating and validating TALENs, TALE-TFs and other TAL effectors-based targeted genomic modification tools.

Genome-CRISP™ CRISPR-Cas9 products and services – GeneCopoeia offers CRISPR-Cas9 sgRNA design and cloning services, Cas9 expression clones and other related products and services for simple and fast targeted genomic editing.

 

Cas9 stable cell lines

Genome-CRISP™ CRISPR-Cas9 stable cell lines – GeneCopoeia offers stable cell lines constitutively expressing the CRISPR Cas9 nuclease for a convenient means to carry out CRISPR genome editing applications with high efficiency, especially for the highthoroughput sgRNA library screening.

Resources

Product Datasheets

Download Product Datasheets (Sales Order # is required).

Application Notes

GeneCopoeia genome editing tools for safe harbor integration in mice and humans

FAQs

FAQs about Safe Harbor gene targeting kits and clones and other genome editing products

Webinars

Watch recorded webinar: Solutions for genome editing: TALEN & CRISPR-Cas9, and Safe Harbor Knock-in ORF clones

webinar PPT slides

User Manual

Download User Manual

 
 
 
 

* Promotion is not valid for gene synthesis clones and custom services.