Title

Title: Characterization of a Clr-b-KO mouse mammary adenocarcinoma cell line

Standing

Undergraduate

Type of Proposal

Oral Research Presentation

Faculty

Faculty of Science

Faculty Sponsor

Dr. Munir Rahim

Abstract/Description of Original Work

Background: E0771 is a mouse mammary adenocarcinoma cell line derived from a spontaneous mammary tumour in C57BL/6J mice. These cells represent an excellent model to study immune responses against mammary tumors. The research in our lab is focused on the role of NKR-P1B receptor in anti-cancer immune responses. NKR-P1B is an inhibitory immune receptor that binds to its ligand Clr-b to regulate immune responses. E0771 cells express Clr-b. Our lab disrupted Clr-b gene in E0771 cells using CRISPR-Cas9 gene targeting system. The E0771 Clr-b knockout (KO) cells will be used to study the role of NKR-P1B:Clr-b interactions in immune responses against mammary tumors in mice.

Purpose: The objective of my work is to characterize the mutations introduced by CRISPR-Cas9 system in the Clr-b gene in the E0771 Clr-b KO cell line.

Methods: Using RT-PCR technique, Clr-b cDNA is amplified from E0771 and E0771 Clr-b-KO cell lines, cloned into a plasmid vector, and sequenced.

Results: Clr-b mRNA was detected in eleven single-cell clones of E0771 Clr-b KO cells by RT-PCR. Clr-b cDNA was cloned into pCR2.1 vector and transformed into E. coli bacteria. After selection and growth of transformed bacteria, plasmids were purified. Sequences of the mutant Clr-b cDNAs will be determined and compared to wild type Clr-b sequences to identify mutations that have disrupted Clr-b gene expression in the KO cells.

Conclusion: This work will aid in the selection of clones of the KO cell line that can be used to study NKR-P1B:Clr-b interactions in anti-tumor immune responses.

Availability

I am available anytime from March 29th-April 1st.

Special Considerations

Rwan Galaleldin Taha (galalelr@uwindsor.ca)

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Title: Characterization of a Clr-b-KO mouse mammary adenocarcinoma cell line

Background: E0771 is a mouse mammary adenocarcinoma cell line derived from a spontaneous mammary tumour in C57BL/6J mice. These cells represent an excellent model to study immune responses against mammary tumors. The research in our lab is focused on the role of NKR-P1B receptor in anti-cancer immune responses. NKR-P1B is an inhibitory immune receptor that binds to its ligand Clr-b to regulate immune responses. E0771 cells express Clr-b. Our lab disrupted Clr-b gene in E0771 cells using CRISPR-Cas9 gene targeting system. The E0771 Clr-b knockout (KO) cells will be used to study the role of NKR-P1B:Clr-b interactions in immune responses against mammary tumors in mice.

Purpose: The objective of my work is to characterize the mutations introduced by CRISPR-Cas9 system in the Clr-b gene in the E0771 Clr-b KO cell line.

Methods: Using RT-PCR technique, Clr-b cDNA is amplified from E0771 and E0771 Clr-b-KO cell lines, cloned into a plasmid vector, and sequenced.

Results: Clr-b mRNA was detected in eleven single-cell clones of E0771 Clr-b KO cells by RT-PCR. Clr-b cDNA was cloned into pCR2.1 vector and transformed into E. coli bacteria. After selection and growth of transformed bacteria, plasmids were purified. Sequences of the mutant Clr-b cDNAs will be determined and compared to wild type Clr-b sequences to identify mutations that have disrupted Clr-b gene expression in the KO cells.

Conclusion: This work will aid in the selection of clones of the KO cell line that can be used to study NKR-P1B:Clr-b interactions in anti-tumor immune responses.