Penn Gene Targeting Service

The Penn Gene Targeting Services 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).

In the past three years (2011-2013) 49 total mouse projects (of these ~15 in the C57BL6 background) were completed. Of these projects 46 went germ line and the remaining 3 did not go germ linebecause of definite orlikely embryonic lethality.

Examples of publications describing targeted mice generated by the Penn Gene Targeting Core and Laboratory are listed here. Publications detailing the expertise of the Core regarding stem cells and regulation of gene expression are shown here.

GET A QUOTE:

For researchers of the University of Pennsylvania and affiliated institutions, prices are about one third to one half below those of comparable commercial services. In fact, even for outside academic researchers prices are still usually below those of the average commercial services. Although we have set prices for specific services, overall charges for a project are strongly dependent on the individual nature of the project and therefore its best to get a quote. We also give volume discounts for some services.

Main Services (offered individually or together)

For further details see here.

1


Establishment of molecular map of BAC-derived targeting vectors or synthetic DNA - based targeting vectors
that are tailor made for the desired mutation and based on a preexisting basic biological design.

2a


Construction of BAC-derived targeting vectors or synthetic DNA-based targeting vectors
either at the Penn Gene Targeting Core, or at the DNA Sequencing Facility or at outside companies, depending on the project. Even the most complex knockin vectors, such as vectors that insert drug-inducible and tissue-specific transgenes into the ROSA 26 locus, can be generated.

2b


Design and construction of gene specific Cas9-associated guide RNAs.
Based on the pre-existing targeting strategy and the properties of the chosen CRISPR system the best guide RNAs are designed and constructed.

3a

3b


Electroporation of finished targeting vectors and/or guide RNAs
 into one of several 129 or C57BL6 mouse ES cell lines or human ES cell or iPS cell lines and selection with suitable drugs, depending on the vector. Our mouse ES lines exhibit high rates of germline transmission (for details see here).

4


Genomic Southern and genomic PCR screening of any cell type, including targeted ES /IPS lines
, and identification of the correct mutation. Please note that PGT accepts any cell type from which sufficient genomic DNA can be isolated for genomic Southern and PCR genotyping.

5a5b


Chromosome counting of recombinant mouse ES clones and G-banding of human ES cells or IPS cells.
Chromosome counting is done at the Core and G - banding is done in collaboration with outside vendors.

5c


Expansion and preparation of mouse ES cell lines for blastocysts injection.
The Core coordinates timing of the mouse ES cell; expansion with the Transgenic and Chimeric Mouse Facility, which performs the injection into mouse blastocysts.

6


Injection into mouse blastocysts.
Done at the Transgenic and Chimeric Mouse Facility, which performs the injection into mouse blastocysts and produces the chimera.

Affiliations