The Penn Gene Targeting Core and Laboratory provides a truly complete knockout and knockin mouse service. In addition it provides all steps necessary for generation of knockout and knockin mutations in human embryonic stem cells (human ES cells) and human induced pluripotent stem cells (human IPS cells). For overview see Main Services below and for details see here. Depending on each project, PGT is using classical gene targeting methods or the revolutionary CRISPR/Cas9 – based system or a combination of both:
• targeting vector design and construction
• CRISPR/Cas9 design and construction
• electroporation of the targeting vectors and/or CRISPR/Cas9 guide RNAs into mouse ES cells or human ES or IPS cells
• PCR/Southern genotyping and karyotyping of the resulting ES/IPS clones
• targeted mouse ES cell injection into blastocysts (usually together with the Transgenic and Chimeric Mouse Facility)
It also provides general genomic Southern/PCR services for genotyping of mouse tails as well as any cell type from which sufficient genomic DNA can be obtained (human ES cells, human iPS cells, mesenchymal stem cells and others).
"The final price of constructing the targeting vector in the Core will depend on the number and nature of cloning steps. This is usually in the range of $3,000 - $6,000. BAC recombineering is used for construction in the majority of cases to ensure long arms of homology. If outside vendors are used for construction, vendors charges will apply. The decision whether to use outside vendors or the UPenn Core is taken jointly by the client’s lab and PGT after thorough discussion of the technical specifics and optimal timing of the project. In some cases synthesis of up to 3000 nt DNA fragments may be required. PGT has extensive experience using synthetic DNA for vector construction. PGT has extensive experience in the construction of all types of targeting vectors, including those introducing knockin mutations, reporter cassettes, stop cassettes and floxed alleles. In addition, vectors targeting tissue and/or drug-inducible transgenes to specific loci, including the Rosa 26 locus, can be constructed."
IMPORTANT: To create economy of scale, at least 3 to 4 projects will be processed together as a batch order. Projects in a batch order may be from the same lab or from different labs. For each project at least 3 guide RNAs will be designed and constructed, unless otherwise specified.
Assuming 3 to 4 parallel projects (9-12 guide RNAs) in a batch order, the following prices apply:
Design and construction of gene specific Cas9-associated guide RNAs: $300 per guide RNA
Design and construction of donor oligos or of large, complex targeting vectors: Cost depends on type of donor DNA or targeting vector. Classical targeting vectors range from about $2,000 to $6,000. For donor DNA oligos, which are usually at least 150 nt long, total cost is the cost of synthesis at IDT plus a PGT processing fee and usually ranges from $200 to $300. Existing transgene constructs are accepted as basis for generation of targeting vectors, and prices will vary, depending on the length of the homologous arms to be added.
Transfection of one specific guide RNA with or without donor DNA into mouse ES cells and generation of up to 45 ES cell clones by pre-selection with G418 or puromycin: $1,500. For an additional charge, pre-selection by GFP- FACS is also possible. The mouse ES cells can either be used to generate a mouse by blastocyst injection or just to measure effectiveness of a guide RNA.
Transfection of one specific guide RNA with or without donor DNA into human ES cells or human IPS cells and generation of up to 45 clones by pre-selection with puromycin: $2,000. For an additional charge, pre-selection by GFP- FACS is also possible.
Genotyping by genomic PCR, including detection of off-target effects, if necessary. For prices, please see the genotyping services section described under section A) above.
Karyotyping for mouse ES cells is provided by the core as described under section A) above. Karyotyping of human ES cells is done for a flat charge of $700 per clone and includes growing the cells as well as G-banding of chromosomes.
"The chromosome number of a minimum of 10 metaphases is determined by fluorescent microscopy. All images are stored digitally and are available to the client."
"$5,800 for V6.5 ES cells (C57BL6 x 129 Sv), R1 ES cells (129 Sv x 129 SvJ) and TL1 ES cells (129 SvEvTac) or $6,800 for C57B/6 ES cells. We usually pick 192 clones for further screening (from two 96 well plates). Germline transmission is guaranteed for the V6.5 ES line as long as the targeted allele does not cause embryonic lethality. We will not charge additional fees to obtain germline transmission if it cannot be obtained after injection of at least three independent clones. All the above cell lines have been extensively tested for germline transmission: For the V6.5 C57BL6 x 129 Sv hybrid line > 80% of injected ES clones go germline. In addition, PGT has developed improved growth conditions for C57BL6 ES cells. PGT routinely achieves rates of germline transmission for this desirable but technically challenging mouse background that are comparable to the rates for some 129 ES cells (about 55 % for a single C57BL6 ES clone)."
"The molecular map is put together based on the preexisting basic biological design of the vector. For establishment of the molecular map there is a flat fee of $1,500 (this fee also covers final quality control of the finished vector by restriction enzyme analysis and sequencing). The basic biological design of the vector takes into account the structure of the gene and exon(s) to be targeted and the mutation to be introduced (floxed alleles, any type of deletion or knockin mutation)."
"Expansion of homologous recombinant ES clones for injection into blastocysts followed by preparation of single cell suspension for exactly timed delivery to the Transgenic Core. Blastocyst injection of ES clones is performed by the Transgenic and Chimeric Mouse Facility"
"Design and testing of suitable Southern blot probes to detect correct insertion of the targeting vector along its entire length and at least one genomic PCR primer pair to detect the presence of a targeted mutation: Flat price of $1,700 if only one Southern probe and one genomic PCR primer pair are is needed. $2,700 if two working Southern probes (binding just outside of opposite sides of the integrated vector) and one genomic PCR primer pair are required."
"For 40 ES clones: $1,600. Without isolation of genomic DNA: $1,000. Usually we obtain between 5 and 30 initial positive clones. For certain targeted genes targeting efficiency is naturally low due to epigenetic / structural issues at the targeted locus. If an electroporation must be repeated for this reason we will charge only 70% of the original price for the electroporation."