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Services


CRISPR/Cas editing
Conventional Targeting
Cryopreservation and Cryorecovery
Rederivation
Speed Line Expansion/Congenics
Neon® Nucleofection
Consultation and Grant Support

 

CRISPR/Cas editing

Artists rendering of CRISPR Cas9 technology with RNA guide and DNA depicted

Nishimasu et al., Cell 2014

Overview

We use the latest available CRISPR/Cas technologies to generate small modifications such as indel-knockouts, deletions, point mutations and conditional knockouts; as wells as insertion of large knockins such as reporters or exogenous expression cassettes.  Depending on the modification required we will recommend the best targeting approach.  We support all steps of CRISPR projects including:

  • Targeting strategy and design
  • gRNA selection and validation of efficiency
  • Oligo template design and synthesis order
  • Construct design and cloning
  • Zygote microinjection or electroporation with DNA/RNA/Cas9 protein
  • Cell line transfection via lipofection or electroporation.
  • Isolation of edited cells by FACS. Expansion and screening of clonal cell lines.
  • PCR genotyping strategy and optimization, genotyping and validation of GE clones or offspring by T7E1 assay and sequencing

Currently, we support in-house CRISRP/Cas editing for rats, mice, and cell lines.  We provide consultation for editing in zebra fish, mosquito and other species.  Please contact us if you are working with a different experimental system and need assistance.

Service details:

For simple modifications (indel knockout, deletion, point mutation) we use one or two gRNAs, and a donor oligo when a repair template is required.  for targeting and the service fee includes manipulation of 100 embryos or generation of 5 founders, whichever comes first. If more embryos are required to achieve a modification, additional costs may apply.

For cell lines we support FACS sorting, single clone expansion and screening of two 96 well plates per gRNA.

For conditional knockouts we currently require two rounds of targeting to insert LoxP sites successfully on the same allele, if feasible we can also use a construct template to generate a replacement (those will be treated as a large insertion projects). We perform gRNA validation for these projects, to increases chances of correct repair.

For large insertions, we currently generate a template construct with homology arms and use it in conjunction with CRISPR.  As other methods such as HITI become available to increase efficiency of CRISPR-mediated knockins, we will incorporate those into our workflow. We perform gRNA validation for these projects, to increases chances of correct repair.

For generating genome edited mouse lines on a background strain other than C57BL/6, FVB/N, CD1 or rat lines on a strain background other than SAS (Sprague Dawley) or Fischer F344, additional animal costs may apply.

We require that newly generated founder and chimera animals be transferred to the requesting investigator laboratory within two weeks of transfer approval and serology testing (if required).  If you require any additional services to be performed on the animals, per diem charges for animal housing and care by TGEF will be incurred.

 

Conventional Targeting

Close up photo of microinjection of cell in lab setting

Microinjection for conventional transgenic generation.

 

We utilize microinjection to generate models with conventional targeting either by pronuclear injection to generate traditional transgenics or 8-cell/blastocyst ESC injection to generate chimeric mice. We provided the following services:

  • Design and cloning of targeting constructs
  • Pronuclear zygote injection of transgene plasmid DNA
  • Blastocyst/8-cell stage embryo microinjection of ESCs
  • PCR genotyping strategy and optimization, founder genotyping
 Service Details

We support generation of conventional transgenics by pronuclear injection of plasmid DNA.  We offer constructing of transgenic vectors or can directly microinject user-generated constructs. Microinjection of 200 embryos is included in the cost for each project fee.

ES-cell targeting by homologous recombination is also available. A complete project includes cloning of targeting vector, ES cell electroporation and selection, clone picking, screening and expansion. We provide screening by long range PCR for up to 300 clones.

For microinjection of ESCs either for newly targeted lines or from existing cell lines (e.g. KOMP stocks) into blastocysts, the service fee includes expansion and microinjection of up to 3 clones or a total of 150 embryos, or 5 chimeras, whichever comes first.

Genotyping of F1 generation is not included in our service, founders and chimeras will be delivered to the investigator for testing of germline transmission and founder line characterization.  If you require breeding to F1 generation and/or breeding at TGEF facility, additional per diem and labor costs will be incurred.

We require that newly generated founder and chimera animals be transferred to the requesting investigator laboratory within two weeks of transfer approval and serology testing (if required).  If you require any additional services to be performed on the animals, per diem charges for animal housing and care by TGEF will be incurred.

 

Cryopreservation and Cryorecovery

Gloves hands remove samples from cryo freezer

We offer both cryopreservation and cryorecovery services for rats, mice and cell lines. We can cryopreserve fertilized embryos from rats and mice, and cryopreserve sperm from mice. We can revive mice from frozen sperm or embryo archives from worldwide resources.

Cryopreservation of sperm is available for mice, we require that you provide 2-4 adult males (15-24 weeks old) that are proven breeders.  Service includes one freezing session and cryo storage for 3 years.  After the initial 3-year period a storage fee would apply if you would like to continue storing samples with TGEF.

Cryorecovery from sperm is performed via IVF with wild type eggs, and embryo transfer into pseudopregnant recipients. This service is currently only available for mice.  Service fees for one session of cryorecovery includes both IVF and embryo transfer charges. We will provide you with the resultant offspring and you’re responsible for screening and genotyping litters, unless you request additional services from TGEF. Because of the cost for egg donor animals and labor, every cryorecovery attempt is counted as a session. If there are no offspring produced because of sperm quality, service fee will be charged as follows: if there were no fertilized eggs after IVF, only the cost of IVF will be charged; if there were fertilized eggs and embryo transfer surgeries were performed, the full service fee will be charged.

Cryopreservation of fertilized embryos requires the user to provide plugged females that have been superovulated and mated.  The investigator is responsible for superovulation and mating of female embryo donors.  TGEF can assist you with required protocols for superovulation and mating, and provide hormones at no extra cost. Service charge includes 1 session of embryo freezing from at least 3 donor females.  Typically we’d like to freeze down at least 100 fertilized embryos per session.  Service fee includes liquid nitrogen storage for 3 years. After the initial 3-year period a storage fee would apply if you would like to continue storing samples with TGEF. The storage fee will be calculated based on the number of samples stored with us and the real cost for liquid nitrogen from the previous year. This service is currently available for both rats and mice.

Cryorecovery from fertilized embryos requires an embryo transfer of frozen embryos into pseudopregnant recipients.  Service fee for one session includes up to 4 embryo transfer surgeries.

For all Cryorecovery services, we will provide you with the resultant offspring and you’re responsible for screening and genotyping litters, unless you request additional services from TGEF.

Freezing of cell lines
Expansion and freezing of ES cells or other cell lines is also available.  We freeze a minimum of 5 vials per line or clone, liquid nitrogen storage for 3 years is included in the service fee. After the initial 3 year period a storage fee would apply if you would like to continue storing samples with TGEF. The storage fee will be calculated based on the number of samples stored with us and the real cost for liquid nitrogen from the previous year.

Dry Shipper Rental
We offer rental of a liquid nitrogen dry shipper if you require frozen samples to be shipped by your laboratory. Please use the liquid nitrogen dry shipper to import any frozen sperm or embryo samples, and handle them with care.

 

Rederivation

If you need to remove pathogens from your mouse or rat lines, rederivation can be performed by TGEF, so the investigator can import animals into MSU, transfer these animals to a clean facility or send them to other institutes. The investigator will need to provide plugged females as embryo donors that have been superovulated and mated.  TGEF can assist you with required protocols for superovulation and mating. We will also provide hormones to investigators for superovulation for the rederivation line, at no extra cost.

 

Speed Line Expansion/ Congenics

These services are currently only available for mice.  We use generation of fertilized embryos by natural mating or IVF, and embryo transfer into pseudopregnant recipients in order to quickly expand a line or move an existing mutation or variant onto a new genomic background.

For speed congenics, we use IVF to speed up the time between successive generations required to transfer a mutation or variant onto a new genomic background, by collecting embryos from 3 week old females from a line of interest and performing IVF with sperm from a pure background male.

Speed line expansion is performed using the same procedure as speed congenics and is useful if a large cohort of animals is to be generated with the assistance of IVF.

All rates listed are per session, for multiple generations, multiple sessions will be required.

 

Neon® Nucleofection

Photo of front of Neon equipment on lab bench

Electroporation of difficult to transfect eukaryotic cells, can greatly assist the introduction of DNA, RNA or RNP into your cell lines of interest.  Investigators can book the use of the Neon® Nucleofector electroporation system.  A service fee includes use of equipment and consumables for 10 electroporation reactions/samples.

A database of cell types transfected successfully with the Neon system, can be used as a starting point for parameter optimization

http://www.thermofisher.com/us/en/home/life-science/cell-culture/transfection/transfection—selection-misc/neon-transfection-system/neon-protocols-cell-line-data.html

 

Consultation and Grant Support

We provide assistance with molecular and reproductive biology techniques outside the listed services. Specifically, we provide training and consultation on different methods and projects.  If you require assistance with a particular project or an experiment, we charge an hourly rate for the time of a TGEF specialist that will assist you with your project.

We provide assistance with grant submission in the form of letters of support or consultation on experimental and project design related to gene editing, neuroscience or reproductive biology.  We also support grant editing for relevant aims, on a limited basis.