<i>In vivo</i> T cell development with LSK-EVAi cells.

<p>(A, left panel) <i>Rag2/γc</i> mice were transplanted with either LSK cells infected with a non-interfering (LSK-CT) or with an Eva1-interfering lentivirus (LSK-EVAi). After 8 weeks, thymi were excised and compared: thymus from mice transplanted with LSK-EVAi were comparable to untreated ones for dimension and total cellularity (A, right panel). Untr., untreated control animal. Scale bar, 2 mm. (B) Time course of T cell development of LSK-CT (white bars) or LSK-EVAi (black bars) cells respectively, in recostitution experiments in <i>Rag2/γc</i> mice. Flow cytometric analysis of CD25/CD44 and CD4/CD8 stainings revealed that LSK-EVAi reconstituted thymus had a delay of T cell differentiation with an accumulation of DN cells at 6 weeks after trasplant (left panel). Percentage of DN cells in LSK-EVAi reconstituted thymus persists higher than in LSK-CT reconstituted thymi at 8 weeks after transplant (right panel). Mean and SD of three independent experiments are shown (** p<0.01). (C) Counts of total DN cells generated in the three different mouse groups at six and eight weeks post-treatment.</p

Analysis of <i>Eva1</i> expression.

<p><i>Eva1</i> real-time RT-PCR in fetal thymi (A), adult DN subpopulations (B, left panel) and thymocytes from mutant mice (B, right panel). mRNA from flow cytometrically purified TECs (CD45-) and intrathymic haematopoietic cells (CD45+) or DN1-3 subpopulations was reverse transcribed and used as the template for PCR with <i>Eva1</i>-specific primers. All samples were normalized to the geometric mean of the GAPDH housekeeping gene. NB, newborn; 2mth, two months; WT, wild type; ΔCAM, Tg-Calcineurin; <i>Prkdc</i>^scid, protein kinase, DNA-activated, catalytic polypeptide (C, upper panel) Eva1 interference in LSK cells by lentiviral vector was controlled by real time RT-PCR. LSK-CT cells shown a comparable expression of <i>Eva1</i> while LSK-EVAi cells shown a drastic decrease of <i>Eva1</i> expression, indicating that the interference has occurred. (C, lower panel), fluorescence microscopy analysis confirming <i>Eva1</i> interference in LSK-EVAi cells. LSK-WT, uninfected LSK; LSK-CT, LSK infected with a non-interfering lentiviral vector; LSK-EVAi, LSK infected with a <i>Eva1</i>-interfering lentiviral vector.</p