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Breast cancer is a multifaceted illness that is impacted by several cellular and hereditary variables. To better understand the onset and course of breast cancer, mouse models are essential. Conventional models do not target mammary epithelial cells specifically or with temporal control. This protocol describes a unique method for accurate temporal and cell-type-specific breast cancer modeling in mice using intraductal injection of Ad-Cre.
| name | Comments | Related Products and Services |
| Luminal mammary epithelial cells (MECs) | Luminal and basal MECs are the targets of certain promoters (Keratin 8 or Keratin 5) that induce Cre expression in the adenoviral vector for genetic modification. To accomplish cell-specific targeting, Ad-Cre is directly injected into the mammary glands (MGs) of mice. | Human p95HER2 Stable Cell Line-T47D |
| Basal mammary epithelial cells (MECs) | ||
| Epithelial cells of other organs | ||
| Hematopoietic cells | ||
| Ad-Cre (adenovirus expressing Cre recombinase) | To selectively induce genetic modification based on Cre/loxP recombination inside the mammary ductal tree, Ad-Cre is injected into MGs. | Cre adenoviral particles Premade Adenovirus Particles Human Cre-GFP adenoviral particles |
| Trp53 (p53 tumor suppressor gene) | To create breast cancer mice models with particular genetic changes, Cre/loxP recombination-based genetic techniques are used to develop and manage floxed conditional knockout or knock-in mouse lines (e.g., Trp53, Brca1, Rosa26, Pik3ca). Within the mammary gland epithelium, promoters (such as Krt8, Krt5) are used to induce Cre expression in a cell-type-specific way. | Gene Synthesis and Cloning Service miRNA 3'UTR Clones loxP/GFP/RFP (Puromycin) Color-Switch Human BRCA1 ORF Clone |
| Brca1 (Breast cancer 1 gene) | ||
| Rosa26 (Gt(ROSA)26Sor gene) | ||
| Pik3ca (Phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha gene) | ||
| Keratin 8 (Krt8 gene) | ||
| Keratin 5 (Krt5 gene) | ||
| Cre recombinase |
a. Acquisition of Mouse Lines:
- Obtain floxed conditional knockout or knock-in mouse lines relevant to breast cancer, such as Trp53tm1Brn (referred to as Trp53L/L), Brca1tm1Aash (Brca1L/L), or Gt(ROSA)26Sortm1(Pik3ca*H1047R)Egan, from reputable sources.
- Additionally, acquire a conditional Cre-reporter line, such as Gt(ROSA)26Sortm1(EYFP)Cos (referred to as R26Y), to facilitate the tracking of mammary epithelial cells (MECs) undergoing Cre-mediated recombination.
b. Breeding Strategy:
- Mate Trp53L/L homozygous mice with R26Y homozygous reporter mice, or with mice carrying homozygous R26Y reporter alleles along with any additional floxed conditional knockout or knock-in alleles, to produce heterozygous F1 progeny.
- To produce F2 compound female mice homozygous for each allele (as experimental subjects) and R26Y-only homozygous females (as control subjects), intercross heterozygous F1 mice. PCR analysis is used to genotype F2 mice.
c. PCR Genotyping:
- Utilize PCR to ascertain the genotypes of mice by utilizing certain primers and cycle parameters.
- Use primers R26YFP-1, R26YFP-2, and R26YFP-3 for R26Y genotyping, amplifying for 35 cycles at 94 °C.
- For Trp53L/L genotyping, employ primers p53F2-10 1F and p53F2-10 1R, amplifying at 94 °C for 35 cycles.
- Determine the homozygotes, wild-type, and heterozygotes for each allele by analyzing the PCR results.
Note:
R26Y Primers:
- R26YFP-1: AAA GTC GCT CTG AGT TGT TAT
- R26YFP-2: GCG AAG AGT TTG TCC TCA ACC
- R26YFP-3: GGA GCG GGA GAA ATG GAT ATG
Interpretation:
- A single PCR band of 250 bp indicates an R26Y homozygote.
- A single PCR band of 500 bp indicates a wild-type (WT).
- Two PCR bands (R26Y: 250 bp, WT: 500 bp) indicate an R26Y heterozygote.
Trp53 L Primers:
- p53F2-10 1F: CAC AAA AAC AGG TTA AAC CCA G
- p53F2-10 1R: AGC ACA TAG GAG GCA GAG AC
PCR Conditions:
- Initial denaturation at 94 °C for 3 min.
- Denaturation at 94 °C for 30 s, annealing at 60 °C for 30 s, extension at 72 °C for 1 min, repeated for 35 cycles.
- Final extension at 72 °C for 3 min.
- Maintain at 14 °C.
a. Sterilization: The day before the surgery, autoclave every surgical tool.
b. Injection Mixture Preparation: Dilute Ad-Cre in DMEM media in a 1:10 ratio with 0.01 M CaCl2 and bromophenol blue to create the injection mixture.
c. Anesthesia and Analgesia: Meloxicam is subcutaneously administered as an analgesic after isoflurane is used to anesthetize female mice.
d. surgery Site Preparation: Use hair removal cream to expose the nipple surgery site, and then clean with alcohol and iodophors.
a. Aseptic Technique: Keep the operating environment as aseptic as possible.
b. Incision and Visualization: To see the mammary ductal tree, make an incision between the two fourth inguinal mammary glands.
c. Injection Method: Fill a Hamilton syringe with the Ad-Cre injection mixture, then slowly inject it into the nipple while keeping an eye on how the blue dye diffuses throughout the mammary ductal tree.
d. Confirmation and Closure:
- Examine the dye spreading to ensure there is no leaking into the stromal compartment to verify the intraductal injection was successful.
- Use wound clips to seal the surgical wounds.
Figure 1. Overview of the aseptic setup for rodent surgery. (Xiang, D., et al., 2019)
a. After anesthesia, place the mouse on a heating pad to aid in its recuperation.
b. Analgesia and Monitoring: Subcutaneously give meloxicam to the mouse and keep an eye out for any indications of infection at the location of the incision.
b. Removal of Wound Clips: Do this 7–10 days after surgery.
a. Palpation and Measurement: Detect the size of mammary tumors by palpating injected mice twice a week for the growth of the tumors.
b. Euthanasia and Tissue Analysis: Cut down animals who meet experimental objectives and separate tumor tissues from the mammary glands to be examined using a variety of methods, including expression profiling and flow cytometry.
This protocol describes a unique approach to produce highly particular temporal and cell-type specific mice models of breast cancer. Breast tumorigenesis may be studied in a controlled way by using Ad-Cre intraductal to target mammary epithelial cells for genetic modification. This method offers insightful information on the molecular processes that underlie the genesis of breast cancer.
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