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
Proposal
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)
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.