• Origin: The B16-F10 cell line is derived from C57BL/6J murine skin cells. The cell line is a commonly used murine model for melanoma. 
• Background Information: HLA-A belongs to the HLA class I heavy chain paralogues. This class I molecule is a heterodimer consisting of a heavy chain and a light chain (beta-2 microglobulin). The heavy chain is anchored in the membrane. Class I molecules play a central role in the immune system by presenting peptides derived from the endoplasmic reticulum lumen so that they can be recognized by cytotoxic T cells. This KRAS gene, a Kirsten ras oncogene homolog from the mammalian ras gene family, encodes a protein that is a member of the small GTPase superfamily. A single amino acid substitution is responsible for an activating mutation. The transforming protein that results is implicated in various malignancies, including lung adenocarcinoma, mucinous adenoma, ductal carcinoma of the pancreas and colorectal carcinoma. Alternative splicing leads to variants encoding two isoforms that differ in the C-terminal region. 
• Gene targeting strategy: The B2m gene (Exon2 to Exon3) of mouse were replaced by the sequence encompassing the human B2M CDS, HLA-A*1101 gene that included leader sequence, the KRAS*G12D peptide, the α1 and α2 domains ligated to a fragment of the murine H-2Db gene containing the α3, transmembrane and cytoplasmic domains. Human B2M is highly expressed on the surface of B-HLA-A11.1/hKRAS*G12D B16-F10. 
• Tumorigenicity: Confirmed in B-HLA-A11.1 mice. Application: The B-HLA-A11.1/hKRAS*G12D B16-F10 tumor model can be used for preclinical evaluation of cancer vaccines. 
• Notes:

             ➢ Inoculated cell lines can be suspended with RPMI-1640 stock solution.

             ➢ Before implementing the project, it is recommended to perform tumor growth experiments. The recommended cell inoculation amount is 2E5.

             ➢ In the experiment, it is necessary to ensure that the number of animals inoculated subcutaneously is at least 2 times the actual grouping number.

B2M expression analysis in B-HLA-A11.1/hKRAS*G12D B16-F10 by flow cytometry. Single cell suspensions from wild-type B16-F10 and B-HLA-A11.1/hKRAS*G12D B16-F10 #1-G07 cultures were stained with anti-human B2M antibody (Biolegend, 395712).

Subcutaneous tumor growth of B-HLA-A11.1/hKRAS*G12D B16-F10 cells. B-HLA-A11.1/hKRAS*G12D B16-F10 (2×10⁵) and wild-type B16-F10 cells (2×10⁵) were subcutaneously implanted into B-HLA-A11.1 mice (female, 8-week-old, n=6). Tumor volume and body weight were measured twice a week. (A) Average tumor volume. (B) Body weight. Volume was expressed in mm³ using the formula: V=0.5 × long diameter × short diameter². Results indicate that B-HLA-A11.1/hKRAS*G12D B16-F10 cells were able to establish tumors in vivo and can be used for efficacy studies. Values are expressed as mean ± SEM.

B2M expression was evaluated​​ on B-HLA-A11.1/hKRAS*G12D B16-F10 by flow cytometry. ​​These cells​​ were subcutaneously transplanted into B-HLA-A11.1 mice (n=6). At the end of the experiment, tumor cells were harvested and ​​analyzed for human B2M expression​​ by flow cytometry. ​​