CAPÍTULO 2 MARCO TEÓRICO
2.12 Motores de combustión interna
2.12.2 Sistemas principales del motor de combustión interna
CD8a-CIII-12 TRANSGENIC MICE
A. Southern blot analysis of tail DNA isolated from CD8a-CIII-123 (Lane 4) and CD8a-CIII-12 (Lanes 1 to 3) transgenic and C57B1/10 (Lane 5) non-trans genic mice digested w ith Xhol. DNA from digests were fractionated on a
0.8% agarose gel, transferred to nylon m em brane by southern blotting and hybridized w ith a full length CD8a cDNA probe.
B. Structure of the CD8a-CIII-123 and CD8a-CIII-12 transgenes and the pre dicted Xhol (X) fragm ent sizes generated by head to tail integrations.
1 2 3 4 5
B
14 — 8.5 _ 7.2 — 6.4 — 5.7 — 4 .8 — 4 .3 — 3.7 — 2.3 1.9 M A L Xhol I l C D 8 - C 3 - 1 2 X 11.2K b Probe ProbeJJi
17.5K bJJI
M C D 8 - C 3 - 1 2 3 X 17 .5K b Probe ProbeFIGURE 35: EXPRESSION OF CD8a/Lyt-2.2 IN THE PERIPHERY OF
CD8a-CIII-123 TRANSGENIC MICE
Three colour fluorom etric analysis of CD8a-CIII-123 transgenic (A) an d (CBA/CA X C57B1/10)F2 non-transgenic control (B) mice. Lymphocytes were triple stained w ith anti-CD8a/Lyt-2.1, anti-CD4 and transgene specific anti- C D 8a/L yt-2.2 antibodies and resolved by plotting C D 8a/Lyt-2.1 against CD4. CD 8a/Lyt-2.1‘*' and CD4'*' sub-populations (indicated as CD8 and CD4 respectively) were gated as show n in the dot-plots and analyzed for expres sion of the transgenic CD8a/Lyt-2.2 allele in the histogram s show n u n d er neath the corresponding dot-plot.
C. Structure of the CD8a-CIII-123 transgene. A 12kb BaniHl-Scal genomic fra g m e n t (h ig h lig h ted ) c o n ta in in g the th ree C lu ster CIII HSS (Vertical Arrows, num bered 1, 2 and 3) was linked to the 5' of a 5.5kb genomic Hindlll
fragm ent containing the CD8a/Lyt-2.2 allele (unnum bered arrow show ing location of C luster CII HSS CII-3). Exons of the CD8a gene are show n as filled black boxes w ith the transcriptional orientation of the CD8a gene indi cated by a horizontal arrow. Restriction enzyme sites shown are Hindlll (H),
BamHl (B), EcoRV (E) and Seal (S). Hindlll sites w ithin DNasel-HSS Cluster CIII sequences have been omitted
o
DO
Cell Frequency Cell Frequency
0 10 20 30 40 50 0 10 20 30 40 SO 60 70 Ol w
[
0 0-, m- 00 00 00 00o
0 5 10 15 20 25 30 CD4 1 0 ' 10‘ H (O O O 00 ? OIn order to determine w hether the transgenic CD8a allele is regulated properly d u rin g thym ocyte developm ent, thym ocytes isolated from CD8- CIII-123 transgenic and (CBA/Ca x C57B1/10)F2 non-transgenic adult mice w ere stained w ith anti-CD 8a/Lyt-2.1 (allele expressed by C B A /C a mice), anti-C D 4 and anti-C D 8a/L yt-2.2 (allele encoded by CD8a-CIII-123 an d expressed in C57B1/10 mice) antibodies (Figure 36). Figure 36A show s the FACS dot-plot of the CD8a-CIII-123 transgenic mouse thym us analyzed for expression of the endogenous CD8a/Lyt-2.1 and CD4 alleles. Individual thy mocyte subpopulations were gated and analyzed for expression of the trans genic CD8a/Lyt-2.2 allele as show n in the histograms below. Expression of the transgenic CD8a/Lyt-2.2 allele could be detected on 14.5% of the CD4' CD8" DN and 25% of the CD8'*' SP cell subsets b u t w as absent from the CD4‘*’CD8"^ DP and CD4"^ SP cells. This pattern of expression of transgenic CD8, as defined by the Lyt-2.2 specific antibody, differs to that seen in thym o cyte subsets of (CBA/Ca x C57B l/10)p2 non-transgenic control mice which also express the CD8a/Lyt-2.2 allele on the DP cell subset (Figure 36B). It w as observed that only a proportion of T cells and thymocytes express the transgenic CD8a/Lyt-2.2 allele. This was found to be the case for all trans genic mice analyzed (see below) and therefore is not only integral to the CD8- Cni-123 transgene construct itself but appears to be a general property of all the DNasel-HSS CIII containing constructs.
FIGURE 36: EXPRESSION OF CD8a/Lyt-2.2 IN THE THYMUS OF
CD8a-CIII-123 TRANSGENIC MICE
Three colour fluorom etric analysis of CD8a-CIII-123 transgenic (A) and (CBA/CA X C57B1/10)F^ non-transgenic control (B) mice. Thymocytes were triple stained w ith anti-CD8a/Lyt-2.1, anti-CD4 and transgene specific anti- CD8a/L y t-2 .2 antibodies and resolved by plotting C D 8a/Lyt-2.1 against CD4. Individual sub-populations were gated as shown in the dot-plots and analyzed for expression of the transgenic C D 8a/Lyt-2.2 allele in the h is togram s show n underneath the corresponding dot-plot.
C. Structure of the CD8a-CIII-123 transgene. A 12kb BamHl-Scal genomic frag m e n t (h ig h lig h ted ) co n tain in g the th ree C lu ster CIII HSS (Vertical Arrows, num bered 1, 2 and 3) was linked to the 5' of a 5.5kb genomic Hindlll
fragm ent containing the CD 8a/Lyt-2.2 allele (unnum bered arrow show ing location of C luster CII HSS CII-3). Exons of the CD8a gene are show n as filled black boxes w ith the transcriptional orientation of the CD8a gene indi cated by a horizontal arrow. Restriction enzyme sites show n are Hindlll (H),
BamHl (B), EcoRV (E) and Seal (S). Hindlll sites within DNasel-HSS Cluster c m sequences have been omitted
o
0 0Cell Frequency Cell Frequency
0 2 4 6 0 5 10 Ol w
[
0 0- m- 00 00 g 0 0.o
0 2 4 6 8 10 0 5 10 15 CD4 m i s 0) X o CJ1 CD 0 2 4 6 CD4 0# x a m i (Qo
o?
o
CD8a-CIII-12 transgenic mice
The previous experiment indicated that DNasel-HSS cluster CIII, con taining DNasel-HSS 1, 2 and 3, carries sufficient inform ation to direct the expression of a C D 8a/L yt-2.2 transgene to the m atu re CD8'*’ SP T cells. DNasel-HSS Cluster CIII was also found to be capable of directing expression in the CD4"CD8" DN thymocyte compartment, however to a lower extend. In order to further define which of the constituting Cluster CIII DNasel-HSS is responsible for conferring this p attern of expression on the C D 8a/Lyt-2.2 reporter, further deletions of the DNasel-HSS cluster CIII were carried out. This w as accom plished by sequentially rem oving individual DNasel-HSS from the 3 ' end of DNasel-HSS Cluster CIII (see Figure 33).
A construct was prepared by linking a 8.3kb BamHl fragm ent to the CD8a/Lyt-2.2 reporter gene (Figure 33). This BamHl fragm ent w as found to contain the cluster CIII DNasel-HSS 1 and 2 (Figure 25). Three founder trans genic mice w ere generated w ith the CD8-CIII-12 construct and these were identified by DNA hybridisation. PBL of the CD8-CIII-12 mice were stained w ith anti-CD8p, anti-CD4 and anti-CD8a/Lyt-2.2 (transgenic allele) antibod ies and analyzed by FACS. All three founder transgenic mice w ere found to express the transgenic CD8a/Lyt-2.2 allele. Figure 34A shows the southern blot analysis of the mice. Using a CD8a CDNA probe the transgene w as found to be intact in these mice and as judged from the size of the hybridising
Xhol restriction fragment appears to have integrated in a head to tail manner. One mouse, CD8a-CIII-12.2 (Figure 34, Lane 3) was analyzed further.
Figure 37 shows the FACS analysis of lymphocytes isolated from CD8-
Cni-12.2 transgenic (A), and CBA/Ca non-transgenic (B) control mice stained w ith the above antibody combination. CD4 and CD8 SP populations were identified by plotting CD8p against CD4. The resulting T cell populations w ere gated as in d icated in the d o t-p lo t and analyzed for C D 8 a/L y t-2 .2 expression in the histograms show n below the dot-plot. Analysis of the CD8P
FIGURE 37: EXPRESSION OF CD8cx/Lyt-2.2 IN THE PERIPHERY OF
CD8a-CIII-12 TRANSGENIC MICE
T hree co lo u r flu o ro m etric analysis of CD8a-CIII-12 tran sg en ic (A) an d C B A /C a non-transgenic control (B) mice. Lymphocytes were triple stained w ith anti-CD8p, anti-CD4 and transgene specific anti-CD8a/Lyt-2.2 antibod ies and resolved by plotting CD8P against CD4. CD8p"^ and CD4"*" sub-popu lations (indicated as CD8 and CD4 respectively) were gated as show n in the dot-plots and analyzed for expression of the CD8a/Lyt-2.2 allele in the his togram s show n underneath the corresponding dot-plot. Solid lines in the his togram s show s CD8a/Lyt-2.2 expression in CD8a-CII-12 (A) and C B A /C a (B) mice.
C. Structure of the CD8a-Cni-12 transgene. A 8.3kb BamHl genomic frag m ent (highlighted) containing two Cluster CIII HSS (Vertical Arrows, num bered 1 and 2) was linked to the 5' of a 5.5kb genomic HindlU fragm ent con tain in g the C D 8a/L yt-2.2 allele (unnum bered arrow show ing location of Cluster CII HSS CII-3). Exons of the CD8a gene are show n as filled black boxes w ith the transcriptional orientation of the CD8a gene indicated by a horizontal arrow. Restriction enzyme sites shown are HindlU (H), BamHl (B) and EcoRV. H indlll sites w ithin DNasel-HSS C luster CIII sequences have been omitted.