Services | Overview
How to initiate services
Interested parties can contact assistant core director to discuss preliminary approach of intended creation of a mouse model. Primary requirement to generate a mouse model at BCH using mouse core services is the PI must have an approved IACUC and IBC protocol for the desired model. After approval is granted the mouse core will co-ordinate with PI or lab member to submit request form and provide required reagents before scheduling an experiment.
Requirements
- Approved IACUC and IBC protocol for the desired mouse model.
- Submit electronic request form (eForm) for services signed by PI and designated lab member.
- Mouse core will co-ordinate with PI or lab member to collect required reagents before scheduling an experiment.
- A fund number or purchase order number to reimburse fees for requested services.
Do you have a question? Email form to transgenicmouse@childrens.harvard.edu and Mantu.Bhaumik@childrens.harvard.edu
Gene editing service
Transgenic DNA Injection OR CRISPR Injection eForm [Download PDF]
Mouse genome is edited by using CRISPR/Cas9 reagents. User may choose to design guide RNA, donor template/s or use commercial services. We highly recommend using synthetic guide RNA, PAGE purified single strand oligonucleotide donor/s and Cas9 protein. Additionally if double strand donor construct is to be used as a donor template, then DNA must be purified as described below. Typically 2-3 gRNAs are recommended per target site due to variable cleavage efficiency of gRNA in vivo.
Microinjection service
Pronuclear injection
- CRISPR/Cas9 reagents into fertilized embryos to generate founder
- single or multiple gene knockout
- loxp or frt floxed allele using single strand oligo donor nucleotide (ssODN)
- introduce specific point mutation using ssODN
- reporter mice using ds-Donor constructs
- gene targeted founders using ds-Donor constructs
- reporter knock-in into ROSA26 locus
- Transgenic DNA constructs into pronuclei of fertilized embryos (B6, F1, FVB or mutant strains)
- Transgenic BAC DNA constructs into pronuclei of fertilized embryos
Blastocyst injection
- Gene targeted ES cells introduced into the host blastocysts to generate chimeric mice
- Wild type or reporter ES cells introduced into mutant blastocysts to generate chimeric mice
- Imported ES cell lines from KOMP/EUCOMM/IMMRC
- Mouse induced pluripotent stem cells (iPSC) introduced into blastocysts
Gene targeting in mouse ES cells service
ES Microinjection Service eForm [Download PDF]
Gene Targeting vector DNA is constructed by PI and provided to the core to introduce into mES cells by electroporation followed by drug selection. Surviving single clones are picked and grown in 96 wells. One half of replica plates are frozen and the other half is provided for genotyping to identify correct homologous recombination event. After confirmation, positive clones are expanded to prepare frozen stocks and karyotyped for normal chromosome numbers. After verification 2-3 independent clones are recommended for injection into blastocysts to generate chimeric mice. Chimeras are transferred to PI after weaning at P21.
We also offer CRISPR/Cas9 mediated gene targeting in mES cell lines.
Chimeric mice: We have proven experience in generating chimeric mice from various mouse strains by blastocyst injection. We offer C57Bl/6 as standard blastocyst donor strain, however, we provide blastocyst injection using Albino/B6 for additional fees. Imported mES cells must be tested negative for Mycoplasma sp.
Service facts
- Gene targeting via homologous recombination (HR) using mES cells derived from C57Bl/6 (JM8.A3.N1), F1 (v6.5) or 129Sv (J1) strains. Users can choose number of clones to be picked in 96 –well format and screen colonies for successful recombination event.
- Gene targeting in vitro using CRISPR/Cas9 reagents in also offered as an alternative to microinjection into fertilized embryos.
- Expansion of Homologous Recombination identified positive ES clones
- ES cell karyotyping
Transgenic mouse service
Transgenic DNA Injection OR CRISPR Injection eForm [Download PDF]
Users design and create transgenic DNA construct to generate founder mice via pronuclear microinjection. We recommend using Qiagen Endo-Toxin free Maxi-Prep or any equivalent kit to prepare plasmid DNA or use plasmid DNA preparation service offered by the core. Transgenic construct is released from plasmid backbone by restriction enzyme digestion and transgenic DNA is agarose gel purified at the core to ensure quality controlled reagents towards successful generation of founder mice.
We will perform pronuclear microinjection of fertilized embryos at 0.5 day post-coitum with purified transgenic DNA or CRISPR/Cas9 reagents and transfer microinjected embryos to recipient foster females. After ~3 weeks pups are born and tail biopsies provided at P7 or P8 for genotyping. Genotyping results must be provided before weaning at P21. Identified founders transferred to investigator after transfer is approved by ARCH or receiving institution.
We aim to provide at least two founders/transgenic construct or CRISPR project. If two founders cannot be achieved using two sessions of microinjection then we will perform additional sessions for extra fees. cryopreservation
Cryopreservation, IVF and Re-derivation Service
Embryo/Sperm/Cell line Storage Service eForm [Download PDF]
Embryo Cryopreservation Service eForm [Download PDF]
Sperm Cryopreservation Service eForm [Download PDF]
Rederivation Service eForm [Download PDF]
Mouse strain is cryopreserved either by freezing embryos or sperm in high security sterile straws and stored in IMV Cryo-Bio storage system under liquid nitrogen.
Please note cryopreservation service is only available to mouse colonies housed at ARCH facilities.
Embryo cryopreservation: Investigators provide 6-10 fertility tested heterozygous or homozygous males that are used for mating with wild type egg donor females to harvest embryos. If egg donor females from same strain to be used for mating then investigator is responsible to provide egg donor females at 3-4 weeks of age. After desired number of embryos are harvested and frozen – to ascertain recovery is evaluated by thawing embryos and re-implantation into foster mother, pregnancy is allowed to term and is confirmed by live birth.
There are three options offered for embryo cryo-preservation at 8-cell stage in high security sterile straws. Each straw contain 20-30 embryos and stored in IMV Cryo-Bio System Goblets.
1) 100-150 embryos – quick and economic option to archive a strain (Cryo-1)
2) 150-300 embryos – long term storage of a strain with occasional recovery (Cryo-2)
3) 300-450 embryos – large pool of embryos of a strain for storage and if periodic recovery or future distribution is expected (Cryo-3).
Sperm cryopreservation: is also offered to archive mouse strains. However, due to high variability of frozen/thawed sperm among males, recovery is not guaranteed. We require three fertility tested males that are used to harvest sperm and stored in high security sterile straws as described. Typically 5-8 straws/male is frozen. Recovery is tested by IVF using wild type egg donor females and by transferring fertilized embryos into foster mothers followed by live birth.
ES cell derivation: We offer ES cell line derivation from transgenic, gene targeted or any other mouse models. PI provide males and females to harvest blastocysts and establish ES cell lines. Total number of cell lines is dependent on quality and quantity of blastocysts harvested from a particular strain.
Service facts
- Embryos at 8-cell stage frozen and stored under liquid nitrogen
- Sperm cryo-preservation performed by harvesting sperm using 2-3 fertility tested males and cryopreserved sperm stored under liquid nitrogen
- Live pups recovered by in vitro fertilization using frozen-thawed sperm
- IVF using fresh sperm for age/sex/genotype matched cohorts
- Re-derivation of imported mouse strains by embryo transfer or IVF
Supporting services
- Transgenic DNA construct purification, quality and quantity analysis for microinjection or gene targeting
- DNA extraction from ES clones in 96-well to 10 cm plates
- Mouse tail DNA extraction
- Mouse primary embryonic fibroblast treated with Mitomycin-C for mES feeder layer
- Mouse embryo harvest at E7.5- E18.5
- Establish mES cell lines from mutant mouse strains
- Imported sperm or embryo storage in liquid nitrogen tank.
Pricing
If you are a current user, please login to VPN to access pricing. If you are a new user, please contact the Core directly to request services and specific pricing information.