Fig. 3: Rapid generation of Msln-specific TRex mice that lack Msln.
From: Germline T cell receptor exchange results in physiological T cell development and function
a Sequence and target sites of sgRNAs targeting Trac exon 1 or Msln exon 4. b CD3+ EL4 cells were electroporated with Trac sgRNA or Trac and Msln sgRNAs complexed with Cas9 RNP, followed by rAAV-1045 or rAAV-7431. Msln TCR expression was determined by Vβ9. c Schematic for murine zygote engineering. d Junction PCR design to detect TCR integration. 5’ Forward primers are located within the engineered locus to detect the exogenous TCR and in an intron region upstream of the endogenous Trac locus to detect the WT allele. A 3’ Reverse primer was located in Trac. e PCR gel of junction PCR performed with DNA collected from the 15 pups born from 1045 engineered pseudo pregnant B6 females. 100 Kb ladder is located in the middle lane and 500 and 1000 Kb demarcated. Wild type (WT) indicates amplification of the endogenous locus while knock-in (KI) indicates amplification of the exogenous TCR upstream of the Trac. Pink arrows, 1045+/− heterozygous pups. n = 1 experiment. f T cell frequency in circulation (left, gated on live CD45+ cells) and frequency of Vβ9+ CD4 (middle) or CD8 (right) T cells in representative pups from e. g Quantification of f. Each dot is an independent mouse. n = 5 mice per group. Data are mean ± SEM. h Junction PCR from DNA isolated from the 13 pups born from the 7431-zygote engineered mice. 100 Kb ladder is located on left and middle lanes with 500 and 1000 Kb demarcated. Pink arrows indicate 7431+/− pups and red arrows indicate 7431+/+ pups. n = 1 experiment. i T cell frequency in circulation (left, gated on live CD45+ cells) and frequency of CD4 (middle) and CD8 (right) T cells expressing Vβ9 in representative pups from h. j Quantification of i. Each dot is an independent mouse and include n = 13 animals from h. Data are mean ± SEM. k Representative immunofluorescent (IF) tissue staining of Msln (red) and DAPI (blue) in lungs from WT, 1045 Msln−/−, and 7431 Msln−/− mice. Scale bar, 25 μM. Representative of six biologically independent animals.
