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This protocol describes a technique for employing recombinase-mediated cassette exchange (RMCE) to conduct structure-function research in mouse embryonic stem cells (mESCs). This method makes it possible to analyze the many roles that multidomain proteins play. By combining stem cell differentiation, genome engineering, and gene targeting, it offers a useful way to clarify the exact location of proteins in biological processes.
| Name | Comments | Related Products and Services |
| ROSALUC Mice | They are kept on a mixed 129/C57BL6/Swiss background and, because they are compatible with RMCE technology, are useful for crossing with heterozygous KO mice. | Gene Knockout Mouse Generation Conditional Knockout Mouse Conventional Knockout Mouse Point Mutation Mouse Gene Knockout Cell Line Generation Conditional Knockout Cell Line Services Fragment Deleted Stable Cell Line Gene Knockin Mouse Generation CRISPR/Cas9 Knockin Mouse Rosa26 Knockin Mouse |
| Mouse Embryonic Fibroblasts (MEFs, TgN (DR4)1 Jae Strain) | These fibroblasts are inactivated with mitomycin-C and employed as feeder cells for growing embryonic stem cells (ESCs). | |
| Tg(DR4)1Jae/J Mice | MEFs are produced using a mouse strain and are then employed as feeder cells for the cultivation of embryonic stem cells. | |
| R26-iPSC Mice | R26-iPSC mice are used as RMCE-compatible animals. They are kept on a mixed 129/C57BL6/Swiss background and are utilized to produce homozygous KO blastocysts that are compatible with RMCE in combination with heterozygous KO mice. | |
| pRMCE-DV1 | Rescue constructs are RMCE-mediatedly targeted to the R26 gene using a plasmid vector. | pCAG-GFP Cloning Vector |
| Cre-Excised pRMCE-DV1 | The pRMCE-DV1 variant that has been altered and has undergone Cre-mediated excision to produce targeting vectors. | |
| pCAG-FlpE-IRES-Puro-pA | Plasmid vector with a selection marker for transfection and FlpE recombinase for cassette exchange. | |
| Gateway pDONR221 Vector | Rescue constructs were cloned using an entry vector prior to RMCE targeting. | pDONR221 vector |
| GATEWAY pENTR 1A Vector | DNA fragments are cloned using an entry vector prior to recombination. | pENTR-HA pENTR-Gus |
| Mouse Monoclonal Anti-p120ctn Antibody | An antibody that measures the expression of the p120 catenin protein. | Antibody-Producing Cell Lines Development |
| Mouse Monoclonal Anti-Ecadherin Antibody | Antibody that measures the expression of the E-cadherin protein. |
1.1. Breeding and Selection of Mice
a. Breed heterozygous KO mice on a mixed 129/C57BL6/Swiss background, combining them with RMCE-compatible mice (ROSALUC or ROSA26-iPSC mice, for example).
b. To find heterozygous KO mice with an RMCE cassette at the R26 gene, use PCR.
d. To create homozygous KO blastocysts, cross RMCE-compatible, heterozygous KO mice with one another.
1.2. Blastocyst Collection
a. Schedule mating events: Place male and female mice in the same house for the night, and the following morning, look for signs of copulation.
b. Euthanasia: When euthanizing a pregnant woman, use a technique that has been approved (such as cervical dislocation).
c. Isolating blastocysts: Create a midventral incision, separate the oviduct and uterus, and then use M2 medium to flush the blastocysts out of the uterus.
1.3. Isolation of RMCE-Compatible KO mESCs
a. Set up culture plates: Add DR4 MEFs that have been treated with mitomycin C and coat with 0.1% gelatin.
b. Plastocyst plating: Transfer blastocysts in SR ES cell media supplemented with pluripotin onto gelatinized plates containing MEFs.
b. Medium maintenance: To encourage cell development, replace the medium every two to three days.
d. Outgrowth selection: Select individual ICM outgrowths and separate them into single cells after ten to twelve days.
e. Cell plating: Grow the established mESC lines by plating dissociated cells onto MEF plates that have been treated with mitomycin C.
f. Identification: To find homozygous KO mESCs that are compatible with RMCE, use PCR.
Figure 1. RMCE-compatible KO mESCs Isolation (A) Strategy for breeding to acquire homozygous, RMCE-compatible KO mESCs. (B) Diagram showing how to isolate mESCs utilizing 2i- or pluripotin-based techniques. Bar for white scale: 25 µm. 100 µm is the black scale bar. (Pieters, T., et al., 2017)
2.1. Cloning Rescue Constructs
a. Create primers with AttB tags for cDNA amplification rescue.
b. Amplification: Rescue cDNA with AttB-flanked ends can be amplified using PCR.
c. BP reaction: Use a BP reaction to transfer the rescue cDNA into donor vectors that are compatible with recombination.
d. LR reaction: To create the targeting vector and carry out an LR reaction, use an enzyme mixture of LR clonase.
2.2. Transformation and Validation
a. Bacterial transformation: Convert competent E. coli bacteria from LR reaction mixtures
b. Choose colonies with the appropriate targeting vector in them.
c. Validation: Use restriction enzyme digests, sequencing, and PCR with particular primers to validate the vectors.
3.1. Transfection of mESCs
a. Cell passage: Prepare for transfection by passage KO mESCs that are compatible with RMCE on MEFs.
b. Transfection: Use a Cre-excised pRMCE-DV1 targeting vector carrying rescue cDNA to transfect mESCs.
c. Splitting: DR4 MEFs are layered confluently on culture dishes containing transfected mESCs.
3.2. Confirmation of Correct Clones
a. Selection: Using G418, choose mESC clones that have the proper cassette exchange.
b. Colony selection: Select colonies that surface seven to ten days following G418 selection.
c. Validation: Use PCR to verify the right clones.
Figure 2. RMCE-based Targeting in KO mESCs. (A) Recombinase-mediated assembly of RMCE-compatible targeting vectors. (B) Targeting of potential rescue constructs to the R26 locus of homozygous KO mESCs through RMCE. (Pieters, T., et al., 2017)
4.1. Culture of Targeted mESCs
a. To eliminate MEFs, passage KO mESCs with R26-driven rescue structures first on MEFs and subsequently onto gelatinized plates.
4.2. Formation of EBs
a. Dissociated mESCs should be plated onto non-adherent petri plates to facilitate the production of EBs.
b. Medium maintenance: or encourage EB development, refresh the medium every two or three days.
Studies of structure and function are essential to the study of biology. This methodology effectively clarifies the roles of multidomain proteins by utilizing mESCs and RMCE technology. It provides a fresh method for precisely determining the important structural regions of proteins and how they affect cellular activity.
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