Chapter 2: Material and methods
2.1. Materials
2.1.3. Experimental models
cocktail Hall Institute – Antibody Facility
RB6-8C5 (1:100) CD8 enrichment cocktail
The Walter and Eliza Hall Institute –
Antibody Facility
M1/70 (1:50) CD8 enrichment
cocktail
The Walter and Eliza Hall Institute –
Antibody Facility
F4/80 (1:50) CD8 enrichment
cocktail
The Walter and Eliza Hall Institute –
Antibody Facility
(‘proliferative’ phenotype) that has a mutation leading to a clinically relevant oncogene (CDK4R24C).
Mouse: HC.PmelKO
HCmel12 Pmel knockout clone 17 (generated by Debby van den Boorn-Konijnenberg)
Melanoma cell line that has above mentioned characteristics and a genetic ablation of the gene gp100;
therefore, murine gp100 (low affinity epitope) can be substituted with human gp100 (high affinity epitope).
Mouse: HC.PmelKO.TYRP1-mNeon
Generated during this study
Melanoma cell line that has above mentioned characteristics.
Endogenous TYRP1 protein (example of a MDA) was tagged with mNeon (control cell line).
Mouse: HC.PmelKO.TYRP1-NFhgp100
Generated during this study
Melanoma cell line that has above mentioned characteristics.
Endogenous TYRP1 protein (example of a MDA) was tagged with mNeon plus human gp100. This allowed targeting of TYRP1 by epitope-standardised ACT protocol in vivo.
Mouse: HC.PmelKO.CDK4R24C -mNeon
Generated during this study
Melanoma cell line that has above mentioned characteristics.
Endogenous CDK4R24C protein (example of an oncogene) was tagged with mNeon (control cell line).
Mouse: HC.PmelKO.CDK4R24C -NFhgp100
Generated during this study
Melanoma cell line that has above mentioned characteristics.
Endogenous CDK4R24C protein (example of an oncogene) was tagged with mNeon plus human gp100. This allowed targeting of CDK4R24C by epitope-standardised ACT protocol in vivo.
Mouse: HC.PmelKO.DCT-mNeon
Generated during this study
Melanoma cell line that has above mentioned characteristics.
Endogenous DCT protein (example of a MDA) was tagged with mNeon (control cell line).
Mouse: HC.PmelKO.DCT-NFhgp100
Generated during this study
Melanoma cell line that has above mentioned characteristics.
Endogenous DCT protein (example of a MDA) was tagged with mNeon plus human gp100. This allowed targeting of DCT by TCRtg T cells during in vitro proof-of-principle experiments.
Mouse: HC.PmelKO.GPNMB-mNeon
Generated during this study
Melanoma cell line that has above mentioned characteristics.
Endogenous GPNMB protein (example of a MDA) was tagged with mNeon (control cell line).
Mouse: HC.PmelKO.GPNMB-NFhgp100
Generated during this study
Melanoma cell line that has above mentioned characteristics.
Endogenous GPNMB protein (example of a MDA) was tagged with mNeon plus human gp100. This allowed targeting of GPNMB by TCRtg T cells during in vitro proof-of-principle experiments.
Mouse: HC.PmelKO.SOX10-mNeon
Generated during this study
Melanoma cell line that has above mentioned characteristics.
Endogenous SOX10 protein (example of a transcription) was tagged with mNeon (control cell line).
Mouse: HC.PmelKO.SOX10-NFhgp100
Generated during this study
Melanoma cell line that has above mentioned characteristics.
Endogenous SOX10 protein (example of a transcription) was tagged with mNeon plus human gp100. This allowed targeting of SOX10 by TCRtg T cells during in vitro proof-of-principle experiments.
Mouse: HC.PmelKO.TYRP1-mScarlet
Generated during this study
Melanoma cell line that has above mentioned characteristics.
Endogenous TYRP1 protein (example of a MDA) was tagged with mScarlet (control cell line).
Mouse: HC.PmelKO.TYRP1-SFmgp100
Generated during this study
Melanoma cell line that has above mentioned characteristics.
Endogenous TYRP1 protein (example of a MDA) was tagged with mScarlet
plus murine gp100. This allowed targeting of TYRP1 by TCRtg T cells during in vitro proof-of-principle experiments that compared the impact of epitope affinity on T cell activation.
Mouse: HC.PmelKO.TYRP1-SFhgp100
Generated during this study
Melanoma cell line that has above mentioned characteristics.
Endogenous TYRP1 protein (example of a MDA) was tagged with mScarlet plus murine gp100. This allowed targeting of TYRP1 by TCRtg T cells during in vitro proof-of-principle experiments that compared the impact of epitope affinity on T cell activation.
Mouse: B16-F1 (B16)
ATCC (CRL-6323)
Differentiated melanoma cell line regularly used for in vivo melanoma mouse studies.
Mouse: B16.gB
Generated in the laboratory of Jason Waithman (Park et al., 2019)
B16-F1 expressing full length HSV-1 glycoprotein B. This cell line was in vivo to generate tumour that were analysed via histological methods.
Mouse: B16.TyrKO
B16-F1 Tyrosinase knockout clone D8 (generated by Debby van den Boorn-Konijnenberg)
Melanoma cell line that has a genetic ablation of the gene tyrosinase. This cell line does not produce pigment and is suitable for intravital imaging
studies.
Mouse: B16.ACTB-mNeon
Generated during this study
B16.TyrKO melanoma cell line in which endogenous ACTB protein (housekeeping gene) was tagged with mNeon (control cell line).
Mouse: B16.ACTB-NFgB
Generated during this study
B16.TyrKO melanoma cell line in which endogenous ACTB protein (housekeeping gene) was tagged with mNeon plus HSV-1 gB. This allowed targeting of the cell line by TCRtg T cells during in vitro proof-of-principle experiments.
Mouse: B16.ACTB-mScarlet
Generated during this study
B16.TyrKO melanoma cell line in which endogenous ACTB protein (housekeeping gene) was tagged with mScarlet (control cell line).
Mouse: B16.ACTB-SFgB
Generated during this study
B16.TyrKO melanoma cell line in which endogenous ACTB protein (housekeeping gene) was tagged with mNeon plus HSV-1 gB. This allowed targeting of the cell line by TCRtg T cells during in vitro proof-of-principle experiments.
Mouse: B16.ACTG1-mNeon
Generated during this study
B16.TyrKO melanoma cell line in which endogenous ACTG1 protein (housekeeping gene) was tagged with mNeon (control cell line).
Mouse: B16.ACTG1-NFgB
Generated during this study
B16.TyrKO melanoma cell line in which endogenous ACTG1 protein (housekeeping gene) was tagged with mNeon plus HSV-1 gB. This allowed targeting of the cell line by TCRtg T cells during in vitro proof-of-principle experiments.
Mouse: B16.SWITCHgB Clone 1G9
Generated during this study
Monoclonal B16.TyrKO melanoma cell line in which endogenous ACTG1 protein (housekeeping gene) was tagged with mNeon plus HSV-1 gB.
This allowed targeting of the cell line by TCRtg T cells during in vitro proof-of-principle experiments.
Mouse: B16.SWITCHgB Clone 1H9
Generated during this study
Monoclonal B16.TyrKO melanoma cell line in which endogenous ACTG1 protein (housekeeping gene) was tagged with mNeon plus HSV-1 gB.
This allowed targeting of the cell line by TCRtg T cells during in vitro proof-of-principle experiments and in vivo studies.
Mouse: B16.SWITCHgB Clone 2C3
Generated during this study
Monoclonal B16.TyrKO melanoma cell line in which endogenous ACTG1 protein (housekeeping gene) was tagged with mNeon plus HSV-1 gB.
This allowed targeting of the cell line by TCRtg T cells during in vitro
proof-of-principle experiments.
Human: HEK293T
ATCC (CRL-3216)
Human embryonic kidney cells used for the generation of retroviral particles for transduction.
Table 2.4: Overview of the experimental organisms and strains used in this study
The table shows the experimental organisms/strains and the origin/source.
Organisms / Strains Origin / Source Mouse: C57BL/6J (H2-Db) Janvier LABS Mouse: C57BL/6J (H2-Db) Charles River
Mouse: C57BL/6J (H2-Db) The Peter Doherty Institute for Infection and Immunity
Mouse: pmel-1 TCRtg / Thy1.1 Jackson Laboratory
Mouse: gBT-I TCRtg / Ly5.1 The Peter Doherty Institute for Infection and Immunity
Mouse: OT-I TCRtg / Ly5.1 The Peter Doherty Institute for Infection and Immunity
Mouse: Rag1-/- The Peter Doherty Institute for Infection and Immunity
Mouse: Rag2-/-; IL2rg-/- The Peter Doherty Institute for Infection and Immunity