Service to confirm pluripotency of human and mouse ESC and iPSC lines by Teratoma Formation Analysisafter injection of cells into an animal.

Features:

• - Turnaround time: 4 weeks (Mouse), 7 weeks (Human)
• - Number of cells required / injection site: Mouse ESCs/iPSCs = 0.5 x 10^6, Human ESCs/iPSCs = 1 x 10^6
• - Enhanced experimental protocol: Injection under both the kidney capsule and in testis results in higher efficiency of distinct teratoma formations with differentiated tissues derived from all three germ layers.

Service Includes:

• - Comprehensive report with histological analysis and high resolution publication-grade images of endoderm, mesoderm and ectoderm formation.
• - Tissue blocks and H&E stained tissue section slides

Notes:

• - Frozen sample culture 
• - Pathogen tests (please provide mycoplasma test result showing negative; otherwise additional testing fee will be required.) 
• - Kidney capsule tumor and testis tumor from each mouse will be embedded in one block. Separate blocks are available upon request.
• - Frozen tissue blocks are available.

Technical Details:

• Experimental setup: A total of 4 mice (3 experimental and 1 control) will be used.
• Injection site:Each recipient mouse will be injected at two locations: in the kidney capsule and in the testis.
• Service includes:Cell injection, teratoma harvesting, tissue preparation (embedding and sectioning), H&E staining and histology analysis of teratoma tissue sections.

Human and Mouse ESC/iPSC Characterization in vivo & in vitro

• Teratoma Formation Analysis Service
• EB Formation and Analysis Service
• Pluripotency Marker Characterization (Human / Mouse)
• Karyotyping (Human / Mouse)
• Germline Transmission of mouse ESCs
• Sex determination

We make the line for you, and we characterize it, too!

We offer a wide range of services to help you identify and characterize stem cell lines. 

We have successfully derived many induced pluripotent stem cell (iPSC) lines from patient specific fibroblasts.

We have three methods for iPSC generation: mRNA, episomal (both genomic footprint-free), or retroviral.

Genomic footprint-free Induced Pluripotent Stem Cell (IPSC) Generation Service:

• - No genomic footprint - the non-integrating system is safe in drug discovery and cell therapy applications.
• - Two methods available: Episomal DNA vectors containing the reprogramming factors or mRNA.
• - High reprogramming efficiency (0.2%-0.5%).
• - The reprogramming vectors/mRNA are removed naturally during the cell cycle.
• - Safe to handle - no viral particles.

Targatt offers a mouse embryonic stem cell (ESC) derivation service from any strain of your interest. We can also derive ESC lines from transgenic mice.

Cell line gene modification is a versatile genetic tool for studying gene function, designing diseases models, biopharmaceutical research, drug discovery and many other applications.

Gene targeting is primarily performed in embryonic stem (ES) cells for the purpose of generating knock-out mice. However, targeted gene modification in cultured cell lines can be a considerably easier and more rapid approach of creating valuable research models. Customized gene-targeted mammalian cell lines are a cheap and convenient model system that can be used for many applications in research and drug discovery, including gene function studies and high-throughput screening.

With strong expertise in genetic modification, gene targeting and cell culture, Targatt can help you to make your customized gene modified cell line. We specialized in both adherent and suspension cell culture. We have many traditional mammalian cell lines in stock and we can also adapt our established targeting techniques to less commonly used cell lines. With a great variety of tools for cell line gene modification in place we have successfully generated gene-targeted clonal lines even from hard-to-transfect cells.

Available Gene Modification Strategies (selected):

• Knock-in/knock-out
• Gene replacement
• Gene editing
• Gene therapy
• Mutagenesis

Each project is individually devised and realized. Our service includes an initial project assessment based on your model requirements. A dedicated PhD level project manager will discuss available gene targeting methods, vector design strategies and special culture requirements with you. After targeting we will clonally expand and cryopreserve several stable transgenic cell lines. Upon request we can expand the clones further on a large scale for high-throughput assays.

Applications (selected)

• - Recombinant protein production in CHO cells
• - Disease model in human cell lines for drug discovery
• - Gene therapy in diseased cell line
• - Generation of a specific cell line with TARGATT docking site as a master cell line for site-specific gene knock-in reporter cell line for gene function studies

Targatt also offers additional services wtihin our cell line models line including TARGATT Gene Insertion.

a) NCSCs forming characteristic “lacunae” during
proliferation on ASC’s NCSC media (10X)
b) NCSCs at a cell density ready for passaging.(20X)

Background: One of the defining characteristics of vertebrate development is the migration of neural crest cells from the neural tube. Neural crest cells give rise to a variety of tissues such as melanocytes, some heart vessels, cephalic neuroendocrine organs, connective tissue of the craniofacial skeleton and the neurons and glia of both the sensory and autonomic ganglia of the peripheral nervous systems. The study of neural stem cells and their progenitors, Neural Crest Stem Cells (NCSCs), have direct medical implications. Numerous pathologies, such as skeletal and nervous system disorders and peripheral neuropathies, arise from aberrant migration, specification or differentiation of neural crest cells. In order to assist in your neural crest cell studies, our NCSC media has been optimized to contain all the factors required to support NCSCs plated on fibronectin coated tissue culture surfaces.
Product description: Add 2 mL of NCSC medium supplement to 498 mL of NCSC medium to produce 500 mL of NCSC media.
Storage conditions for NCSC medium: -80°C; 1 year, 4°C; two weeks. Minimize exposure to direct light.
Storage condition for NCSC supplement: -80°C; 1 year, 4°C; two weeks.
Storage condition for NCSC media: -80°C; 1 year, 4°C; two weeks. Minimize exposure to direct light.
Shipping: On dry ice.
Recommended procedure:
1. Thaw NCSC medium either overnight at 4°C or for several hours at RT.
2. Thaw the NCSC medium supplement at RT.
3. Using sterile technique, add the NCSC medium supplement to the NSCS medium. Mix thoroughly and store the newly made media at 4°C for up to two weeks. NOTE: NCSC medium can be filter sterilized if desired. Do NOT filter sterilize the NCSC medium supplement. Add this supplement directly to the NCSC medium.
4. Prior to use, warm the media to RT in the tissue culture hood. Due to the complex composition of the NCSC media, we do not recommend warming the media in a 37 C water bath.

 Price: 80 EUR

Based on our proprietary TARGATT Technology for site-specific knock-in mouse generation, we developed a TARGATT system specifically for introducing gene(s) of interest into mammalian cell lines. The unique advantage of this system is that any gene of interest can be inserted efficiently into a defined, transcriptionally-active locus with high gene expression in a single copy fashion.

 TARGATT Features:

• Site-specific insertion of any gene 
• High integration efficiency
• Single-copy integration
• Stable expression
• Fast: get your transgenic cell line in 3 months!

Targatt proprietary TARGATT Technology enables highly efficient site-specific gene integration in mammalian cells and animals. This technology uses PhiC31 integrase to insert any gene of interest into a specific docking site that was pre-engineered into an intergenic and transcriptionally active genomic locus. Our TARGATT Technology improves several aspects in the generation of transgenic cell lines and animals:

1. High integration efficiency mediated by  PhiC31 integrase reduces time and cost
2. Site-specific integration at a pre-selected genomic locus eliminates position effect and ensures high expression levels of the transgene
3. Integration at intergenic region ensures that no internal genes are interrupted
4. Single copy gene integration eliminates repeat-induced gene silencing and genomic instability
5. Site-specific integration allows a precise comparison of the effects of the transgenes among different lines.

TARGATT Technology can be utilized for a variety of applications including reporter gene expression, gene knockdown and disease models.

Targatt provides a complete range of gene targeting services in rats to generate transgenic knock-in and knock-out rat models.

Rats are more versatile research model animals than mice since they are physiologically more similar to humans. The recent isolation of rat embryonic stem (ES) cells opened the doors to the generation of transgenic rats and we are proud to be one of the first providers of an all-inclusive transgenic rat generation service. Our competence in gene-targeting vector design coupled with our expertise in culturing rat ES cells allows us to offer a high-quality customized service for the generation of transgenic knock-in and knock-out rats.

Using our proprietary germline competent rat ES cell lines we can create your customized transgenic rat model, including

• - Constitutive knockin/out
• - Conditional knockin/out
• - Inducible knockin/out
• - Point mutations
• - Random integration
• - Insertion of reporter genes
• - Humanized rat models
• - Disease rat models
• 

We take great pride in offering you the highest level of service and we are committed to ensuring the success of your transgenic rat project. Our service includes an initial project assessment based on your model requirements. A dedicated PhD level project manager will discuss available gene targeting vector design strategies and will update you continuously throughout the project.

We provide the most flexible customized service options whether you’d like us to do all the work or are on a tight budget and prefer doing some steps yourself.

 Full Transgenic Rat Generation Service Includes:

• - Gene targeting vector design, construction and sequencing
• - Rat ES cell targeting, screening and expansion of positive rat ES cell clones
• - Karyotyping of targeted rat ES cells
• - Chimera production via blastcyst injection
• - Chimera breeding for germline transmission
• - Genotyping of offspring
• - Transfer of heterozygous targeted rats to the customer
• - Optional: breeding to generate homozygous rats

All our animals are housed in a facility that is accredited by the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC) International, has an Assurance on file with the Office of Laboratory Animal Welfare (OLAW), and is a registered research facility with the U.S. Department of Agriculture.

We also offer Knock-in/Knock-out Mouse and Transgenic Mouse (Random Insertion) services. For more information please contact us.

We provide a service to generate transgenic mice by traditional pronuclear microinjection. Using this method, transgenes are inserted randomly into the genome.

Expression of the transgene and the subsequent transgenic mouse phenotype may vary from line to line due to the nature of random gene insertion. We recommend to generate multiple independent trangenic mouse lines in order to interpret the experimental results.

Available mouse strains: FVB, C57BL/6

We also offer Knock-in and Knock-out Mice and Knock-in and Knock-out Rat services. For more information please contact us.

Generation of transgenic mice by random insertion

Step 1: Cloning of cDNA, promoter of interest, and other elements

Step 2: Construction of the transgene vector

Step 3: Pronuclear microinjection of vector DNA into mouse embryos

Step 4: Transfer of injected embryo into recipient mothers

Step 5: Genotyping of offspring to identify potential transgenic founders

Step 6: Breeding to F1 or F2 progenies (optional)

We provide a high quality service for the generation of gene-targeted knock-in and knock-out mouse models. Our team has decades of experience in vector design, ES cell targeting and mouse handling. We will discuss your project needs with you and custom design your knockin/knockout mouse model. Our service includes consultation on available gene targeting vector design strategies and project assessment based on your model requirements.

Service Milestones for the Generation of Knock-in/Knock-out Mouse Models

• - Gene targeting vector construction and sequencing
• - ES cell targeting, screening and expansion of positive ES cell clones
• - Karyotyping of targeted ES cells
• - Cre/FLP recombination (optional)
• - Chimera production by blastocyst injection
• - Breeding of chimeras for germline transmission
• - Genotyping of offspring
• - Transfer of heterozygous targeted mice to the customer
• - Optional: breeding to generate homozygous mice

Features

Cost-effective

• - Targatt has years of experience and an exceptionally high success rate in generating conventional gene-targeted mice with our proprietary mESC lines.

High-Quality

• - Our scientists have extensive experience in gene targeting and genetic mouse models. Our services always include a rigorous quality control with multiple check-points to ensure your projects are completed with highest accuracy. All mice are generated in and shipped from our California facility (NIH guidance certified).

Please contact us with any questions regarding services not listed. We are happy to discuss your project with you and design customized strategies. We also offer Transgenic Mouse (Random Insertion) and Transgenic Rat Services (knockin, knockout).

Testimony of Dr. Zhenheng Guo, PhD, Assistant Professor of Internal Medicine, Division of Endocrinology and Molecular Medicine, University of Kentucky

Project: Germline-transmitted, conditional knock-out mice by tetraploid complementation

"Overall, I am very satisfied with the quality of your service. After we received the mice, we did extensive studies to characterize them. All of the PCR reactions confirm the gene modifications are in the correct sites. We have also crossed mice with Cre mice to demonstrate that the Cre deleted the exon in heterozygous pups as expected. All data obtained showed that the mice were correctly targeted. In addition, I am very happy with the frequent communication with your scientists during the process. I highly recommend your services."

Project types

Knock-in Mouse Models

The introduction of a gene into a specified location of the mouse genome can be used for various applications. Mice can be homozygous or heterozygous for the inserted gene.

• - Reporter genes (e.g. GFP, lacZ) are used for expression analysis of a gene of interest. The reporter gene is inserted into the gene of interested in-frame (non-disruptive), allowing for visualization of temporal and spatial gene expression pattern.
• - Humanized disease models can be generated by inserting a human mutant gene or gene fragment into the corresponding mouse gene. The diseased human allel is thus transcribed in the appropriate genomic context and can be analyzed on behavioral, pathological, cellular and/or molecular level.
• - DNA recombinases (CRE, FLP) can be inserted into a gene of interest. The celltype-specific expression of the recombinase then allows for gene inactivation in the desired tissue after crossing this knock-in mouse with a conditional knockout mouse.

Knock-out Mouse Models

The knockout technology is most commonly used for gene inactivation, either in a constitutive or conditional fashion.

• - Constitutive knock-out mouse models are widely used to study gene function. The gene of interest is permanently inactivated in all cells of the animal. However, this non-conditional knock-out may cause lethality and is therefore not always recommended.
• - Conditional knock-out models are inducible - the targeted gene is excised after crossing the mouse with a Cre-transgenic mouse line. Gene inactivation can be celltype-specific and/or chemically induced.