주요 papers and Vectors

MSGV1
A Novel Subgenomic Murine Leukemia Virus RNA Transcript Results from Alternative Splicing
JOURNAL OF VIROLOGY, Apr. 2000, p. 3709–3714

MSGV1

Created at
Here is a table summarizing the issues related to the development of the MSGV1 vector, as well as relevant details regarding transduction, analysis, and controls:
Aspect
Details
Vector Name
MSGV1 (Murine Stem Cell Virus-based Splice Vector)
Vector Testing Method
Retroviral vector transduction of SupT1 cells
Analysis Technique
Fluorescence-activated cell sorting (FACS) staining for CD3 or anti-Vα12
Vector Integration Confirmation
Confirmed using Southern blot analysis
Cell Line Used for Integration Test
PG13 clone AIB 18
Here is a comprehensive table summarizing the steps and components involved in the construction of the retroviral vectors, as well as the procedures for TCR chain determination, vector assembly, cell transfection, and integration analysis:
Aspect
Details
RNA Isolation & TCR Chain Determination
RNA from MART-1–reactive T-cell clone (M1F12) was subjected to RACE-PCR and DNA sequencing to determine TCRα and β chain usage. TCR usage (IMGT nomenclature): α chain - TRAV35/TRAJ49/TRAC, β chain - TRBV10-3/TRBD1/TRBJ1-5/TRBC1.
Primer Design for Cloning
Primers designed for full-length cDNA cloning based on determined TCR chains.
Poly(A) RNA Isolation
Poly(A) RNA from 1 × 10⁷ M1F12 T cells was isolated using Poly (A) Pure mRNA Purification Kit.
RT-PCR and Cloning
RT-PCR was conducted using Titan One Tube RT-PCR Kit. Amplified TCRα and β chains were gel purified, cloned into pCR2.1-TOPO vector, and confirmed by sequencing.
Vector Backbone
pMSGV1 derived from pMSGV, which uses MSCV LTR. Contains extended gag region and env splice site from SFGtcLucITE4 vector. Modifications for pMSGV1 include Kozak sequence enhancement.
Vector Variants Constructed
Four retroviral vectors (APB, AIB, BPA, BIA) expressing both TCR chains. Each vector constructed with different combinations of PGK promoter, IRES element, and TCRα/β chain orientations.
Transfection of Packaging Cells
PG13 and Phoenix Eco cells cocultured and transfected with vector constructs (AIB, BIA, APB, BPA) using Gene Porter reagent.
PG13 Clone Generation
Clones generated after 14-day coculture and Phoenix Eco cell removal using magnetic antibody-coated beads.
Physical Titer Screening
Initial screening of PG13 clones via RNA dot blot to determine physical titer.
Transduction and FACS Analysis
Highest-titer clones tested via retroviral transduction of SupT1 cells. Analyzed by FACS for CD3 or anti-Vα12 expression.
Southern Blot Analysis
Confirmed vector integration and copy number in PG13 clone AIB 18.
Control Vector
MSGIN vector for GFP expression and anti-gp100 TCR (APB9) vector used for control in transducing PBL or TIL aliquots.
This table provides a structured overview of the experimental steps, from RNA isolation and vector construction to cell transfection and integration confirmation.
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