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To generate a murine Fcgrt-deficient model with a human FCGRT knock-in, a CRISPR/Cas9-assisted gene targeting strategy was used. This involved creating a knockout (KO) of Fcgrt while concurrently inserting a FCGRT mini-gene under the control of the mouse promoter. Cas9, along with gRNA Fcgrt-g91T (protospacer sequence: 5-GGGTCACCCTGTCGGAATG-3), was utilized to introduce the following sequence at the start codon in Fcgrt exon 2: Kozak sequence and human FCGRT cDNA cassette inserted at the cleavage site, Sv40 intron, a 214 bp native FCGRT 3' untranslated region (3UTR), a 152 bp native human sequence 3 of the mapped 3UTR, and a mouse U6 promoter-driven guide RNA scaffold with 2xPolIII terminator (all in reverse orientation immediately 3 of the FCGRT cassette), a splice-trap element with splice acceptor and SV40 polyadenylation sequence followed by 3-frame ATG short open reading frames. This active genetics cassette is designed to trap and truncate any mouse Fcgrt isoforms that may splice past the FCGRT cassette. Additionally, a Flp Recombinase Target (FRT) site is inserted to facilitate Flp-mediated collapse of targeted tandem integration events, if necessary. The investigator responsible for this work notes that the experiments were conducted in mice with a C57BL/6J genetic background.
BioRxiv Doi:10.1101/2022.08.30.505951 ROBUST AND EFFICIENT ACTIVE GENETICS GENE CONVERSION IN THE RAT AND MOUSEChenyen Lai, Oscar Alvarez, Kristen Read, Don van Fossan, Christopher M Conner, Shannon Xaing-Ru Xu, Dale O. Cowley, Valentino Gantz, David R. Webb, Kurt Jarnagin
Colony Surveillance Program and Current Health Reports
Limited quantities of breeder mice (recovered litter) are available from a cryoarchive; recovered litter usually available to ship in 3 to 4 months.
Cryopreserved material may be available upon request, please inquire to mmrrc@med.unc.edu for more information.
Distribution of this strain requires submission of the MMRRC Conditions of Use (COU). A link to the COU web form will be provided via email after an order has been placed; the form should be completed then or the email forwarded to your institutional official for completion.
Additional charges may apply for any special requests. Shipping costs are in addition to the basic distribution/resuscitation fees. Information on shipping costs and any additional charges will be provided by the supplying MMRRC facility.
Some MMRRC strains were submitted by the donor as cryopreserved germplasm, and because these strains were not cryopreserved by the MMRRC, we have not assessed the quality, nor genotype, of this material. Our expertise in reviving mice from various qualities of frozen sperm and embryos, using methods like IVF and ICSI, gives us confidence in successful recoveries in most cases. However, due to the uncertain quality of these samples, we'll limit revival attempts to two per order. Additional attempts are available for a fee, on top of standard charges, if requested. It's important to note that some strains may lack the expected mutation, so we can't assure successful order fulfillment until we attempt to revive the strain.
1 The distribution fee covers the expense of rederiving mice from a live mouse; you will receive the resulting litter. The litter will contain at minimum one mutant carrier; the actual number of animals and the gender and genotype ratios will vary. (Typically, multiple breeder pairs can be established from the recovered litter.) Prior to shipment, the MMRRC will provide information about the animals recovered. If you anticipate or find that you need to request specific genotypes, genders or quantities of mice in excess of what is likely from a resuscitated litter, you may discuss available options and pricing with the supplying MMRRC facility.
2 An aliquot contains a sufficient number of embryos (in one or more vials or straws and based on the transfer success rate of the MMRRC facility) to transfer into one to three recipients. The MMRRC makes no guarantee concerning embryo transfer success experienced in the recipient investigator's laboratory. Neither gender nor genotype ratios are guaranteed.
3 An aliquot is one straw or vial with sufficient sperm to recover at least one litter of mice, as per provided protocols, when performed at the MMRRC facility. The MMRRC makes no guarantee concerning the success of these procedures when performed outside the MMRRC facilities.
4 The distribution fee covers the expense of resuscitating mice from the cryo-archive; you will receive the resulting litter. The litter will contain at minimum one mutant carrier; the actual number of animals and the gender and genotype ratios will vary. (Typically, multiple breeder pairs can be established from the recovered litter.) Prior to shipment, the MMRRC will provide information about the animals recovered. If you anticipate or find that you need to request specific genotypes, genders or quantities of mice in excess of what is likely from a resuscitated litter, you may discuss available options and pricing with the supplying MMRRC facility.