Policies and Guidelines
Need some kind of intro to this section
Policies:
1. Mouse core services are available on a priority basis to all IDDRC investigators. If scheduling permits the core provides services to non-IDDRC and other investigators.
2. To access core services, contact us directly by phone call or email. Provide a description of your mouse model project goal/s in less than 200 words. After reviewing your specific approach we will contact you to discuss best approach in achieving your mouse model.
3. Generation of gene manipulated mouse model using microinjection and micromanipulation techniques rely on various factors – quality of reagents, target site of a gene, biological variation between donor and recipient animals etc. that may contribute to the live births. We make every effort to generate founders with the reagents provided for microinjection, but do not guarantee number of founders.
4. Gene manipulation core only accepts quality-controlled reagents for microinjection. If we determine any reagents fail to meet our QC standard then we reserve the right to request either new reagent/s or not to initiate a project.
5. The mouse colony used by the core is housed within Animal Research Children’s Hospital (ARCH) in an isolated room. The entire colony is monitored by ARCH sentinel program and certified as pathogen free with the exception of Helicobacter sp.
6. Design of appropriate diagnostic genotyping assay to identify founder is of utmost importance for successful completion of a mouse project. Therefore it is necessary to perform genotyping assay that identify correct founder before weaning. If for any reason the mouse colony needs to be weaned at P21 – the investigator is responsible for per diem fees till transfer.
7. Cancellation – experiment is scheduled after receiving completed request form and upon agreement of approximate cost for a specific experiment. If for any reason PI decided to cancel scheduled experiment at the last minute – then the PI will be responsible for the cost of reagents and loss of scheduled work. However, PI may choose to cancel scheduled experiment 15 business days at no cost.
8. The service fees are subjected to change and adjusted according to the cost of materials after annual review. Typically, we estimate fees for a specific service depending on available reagents from vendors. However, final invoice may change depending on the reagents and number of injection sessions needed to complete a project.
9. Payment – An estimated cost is provided at the outset of each service requested. Final service fees are determined after completion of each project. BCH/IDDRC members must provide a cost center number before we can initiate a service. Non-BCH/non-IDDRC entities must have a purchase order number (PO#) before initiating any service. Please check with your administrative office before providing a cost center or a PO number for payments. Note – payment is due at the completion of a project.
10. Service fees are recovered after completion of each project. If core produces live births and no founders are identified then PI is responsible for material cost. However, if unexpected or expected in utero lethal phenotype is identified then full fees will be charged. If core fails to produce any live births then there will be no fees charged to PI.
11. We archive documentation of mouse models generated at the mouse core for future references, especially results of germ line transmission, phenotype and publications. PI must agree to share this information that will be used for mouse core Intellectual and Developmental Disorder Research Center (IDDRC) grant annual report and competitive renewals.
12. Mouse core maintains strict confidentiality of mouse strains generated at the core. We will discuss project related strategies, progress and final outcome with the PI and assigned lab member only – unless the PI agrees to share information with others.
13. Please acknowledge mouse core (IDDRC grant #1U54HD090255) in any publication, oral presentation, poster or other communications.
Guidelines:
Genome editing using CRISPR/Cas9: We recommend choosing 2-3 guide RNA/target sites due to high variability of gRNA efficiency in vivo. In our experience, synthetic gRNA have higher efficiency and purity than in vitro transcribed gRNA. For Knock-In of single stand donor or Megamer must be PAGE purified for successful insertion and post-injection embryo survival. For double strand-donor DNA must be prepared using Qiagen kit described below. We prefer to use crRNA and Cas9 protein and ssODN from IDT. crRNA and ssODN must be delivered to the mouse core as delivered (lyophilized) from IDT.
Transgenic and Gene Targeting construct DNA purification: Prepare plasmid DNA using Qiagen Maxi-Prep Endo-Toxin free kit or use plasmid DNA preparation service provided for a fee. At the last step dissolve DNA in sterile nuclease–free water or 10mM TRIS-pH 8.0 at 0.5-1.0 ug/ul.
If needed, linearize or digest DNA to release transgenic construct from plasmid backbone using 100ug of DNA. Check on an agarose gel for complete digestion with a DNA molecular weight marker. Take picture and deliver digested DNA to the core.
Gene targeting: Traditional gene targeting using targeting vector with homology arms electroporated into ES cells. We offer J1 (129Sv), B6 (JM8) and hybrid V6.5 9129xB6) ES cell lines. After selection ~96 to 192 clones are picked in 96 well format. After frozen stocks are prepared, ES clones in 96-well plates are provided for DNA preparation or ES DNA prepared as a service by the core. After initial genotyping and confirmation, potential positive clones are thawed from frozen 96 well, expended, frozen and prepared DNA. A second genotyping is performed from expanded clones to reconfirm correct targeting event. Confirmed clones are karyotyped to confirm normal chromosome numbers.
If secondary Cre or Flpe transfection is needed, then confirmed ES clones are electroporated using Cre or Flpe plasmid. After selection 24-96 clones are picked and genotyped as describe above.
Chimera production: In house gene targeted 2-3 ES clones with normal chromosome numbers are injected into appropriate blastocysts for chimera production. Imported gene targeted ES clones must be tested pathogen free and normal chromosome numbers to be accepted for blastocyst injection.
Chimeras are graded for extent of chimerism, sexes determined at weaning and transferred to PI’s lab around 4-5 weeks of age. We also offer chimera breeding to test germline transmission as a service.
Mouse colony transfer: Mouse colony transfer from Gene Manipulation core requires approval from ARCH. Approval process includes submission of a completed transfer form and documentation of an approved IACUC protocol in the form of a copy of page/s showing that the strain is approved. We do not accept any live mice once the colony is removed from core mouse room. Mouse core is not responsible for shipping, loss in transit or crating mice for transfer.
Transfer within ARCH: Core will initiate transfer process by submitting a transfer request form and IACUC approved documentation from PI to ARCH Vet’s office. After approval, colony is handed over to the lab personnel. Receiving lab must bring cage cards with IACUC approved protocol number, PI’s name etc. at the time of transfer.
Transfer outside ARCH: Core will initiate an inter-institutional transfer through ARCH export coordinator by submitting an export form. The coordinator will contact receiving institutional Vet’s office and provide required Health report and other documentation. After approval, export coordinator will inform of a specific date/time to arrange for in person pick-up by lab member or a courier service. Mouse core does not deliver mice to any institution.