Insertion of 50 homing guide RNA (hgRNA) loci in locations scattered throughout the genome. This is the MARC1 Founder #3 (PB3) that is generally similar to MMRRC:65424. Each mouse will contain a subset of the inserted homing guide RNA's and the MMRRC will provide a report with each mouse containing the details on the inserted homing guides. Lineage barcoding experiments can be carried out by crossing these mice to a line that expressesCas9 constituitively or conditionally. In progeny of such a cross, hgRNAs begin to mutate themselves in cells that express Cas9 and accumulate heritable mutations.The cells or cell populations under investigation can then be isolated, their hgRNAs amplified and sequenced to obtain their lineage barcodes. Comparison between barcodes of different samples within the same mouse can shed light on the lineage relationship between those samples. Please consult the publication associated with founder line PB7 (PMID:30093604) for further details.
A barcoded library of homing guide RNAs (hgRNAs) with randomized spacer sequences was transposed in v6.5 mESCs using the Piggbac system. Transposed mESCs were selected using puromycin, injected into blastocysts, implanted in surrogate females at the Harvard Genome Modification Core Facility. Among the resulting chimeric mice, males with the highest number of hgRNA integrations were selected as founders of the MARC1 line. One of these founders (chimera #3) carried 50 heterozygote germline-transmissible integrations of hgRNAs. The selected founder was crossed with CD1 females (Charles River 022) to create the F1 generation. In the F1 generation, PCR amplification of the hgRNA amplicon followed by high-throughput sequencing was used to determine the number of hgRNAs inherited to each member. Based on these numbers, pairs of F1 were selected with the objective of maximizing the number of hgRNAs that would be inherited to F2, and crossed to create F2. The same procedure was repeated for F2 and so on to create the following generations. On average, 50 mice were generated per generation, genotyped (by PCR amplification followed by Illumina sequencing), and used to create the next generation. The genetic background of this line is deemed STOCK due to contributions from more than two inbred lines, approx. 50% CD1 (RRID: IMSR_CRL:022), 25% C57BL/6, and 25% 129S4.
Kalhor R, Kalhor K, Mejia L, Leeper K, Graveline A, Mali P, Church GM.Developmental barcoding of whole mouse via homing CRISPR. Science. 2018 Aug31;361(6405). pii: eaat9804. doi: 10.1126/science.aat9804. Epub 2018 Aug 9.(Medline PMID: 30093604)
Leeper K, Kalhor K, Vernet A, Graveline A, Church GM, Mali P, Kalhor R. Lineage barcoding in mice with homing CRISPR. Nat Protoc. 2021 Mar 10. doi: 10.1038/s41596-020-00485-y. Epub ahead of print. (Medline PMID: 33692551)
|Viability and Fertility:||Female||Male||Comments|
|Homozygotes are viable:||Yes||Yes|
|Homozygotes are fertile:||Undetermined||Undetermined|
|Hetero/Hemizygotes are fertile:||Undetermined||Undetermined|
|Age Reproductive Decline:||Undetermined||Undetermined|
Limited quantities of breeder mice (up to 2 males and 2 females or 4 mice) per investigator per month are available from a live colony, usually available to ship in under 12 weeks. Larger quantities may be available, please contact the distributing center directly at firstname.lastname@example.org for more details.
Cryopreserved material may be available upon request, please inquire to email@example.com for more information.
The donor or their institution limits the distribution to non-profit institutions only.
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.
|MMRRC Item #||Description||Distribution Fee / Unit (US $)||Units||Notes|
|Heterozygous / Hemizygous Female
Heterozygous / Hemizygous Male
|$327.00 / Non-Profit||Per Mouse||The firstname.lastname@example.org may assess additional fees for any special requests (e.g., specific age or weight of mice, etc.).|
|Cryopreserved material may be available upon request, please inquire to email@example.com for more information.|
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.