Modelos utilizados en el proceso de fritura

151 tctgtggaat tgagtaggac caagcctgat ttgggtcatg aacatcccca 201 ctctggcagt cctctaggag agctgttcag gggtatgagc agtattagca 251 tttcttctct aatctctgcc tcacagttat ctggctatag ccagggatgc 301 catgactcac tataaagggc tgctttaggg ggttctcata ttctcttc

1 gttcgttccc ctccagcgac tgtcttctta gaatcatgtg actggcagtc 51 taataaccta cctgatacac ttggaagact cctgacccaa tgcagtttct

AP-1/PEA-1

101 tcctaaaatg tcccttacat cccctacccc tgactcaagn aaaataatct 151 agcccttcta tttcaataaa ggagctgtcc ttttaaaaag tcttgcaaat 201 cattcatacc caccatacac actgccccgg aacacgatga tccccagctc 251 ctctgcacag gtctgagacc agagccatgg gggaggggna agtggcattt 301 ggaggaagtc cacttggtta acatgaaacc tttgctatga ctcaaggggn 351 tgttgaggca tatatgtgac cttggttcat acaacagagg tcgttcattc 401 tctgaggagg ggaatgggct tggctaaggt ttgacctatt cagtgtgaca 451 gtcagtagtc aggcttttgg ctgagtagga aggcaggaga gagggctcag 501 tgtgttttta tcttgctagt ggacctctct gcagagggcc tgtcttctct 551 aacacaagcc tggcaacatc tg

Consensus ISRE R G A A A N (N) G A A A C T HS+8.7 ISRE A G A A A C A G A A A C C

to investigating putative factor binding at sites HS+8.7 and HS+8.9, a 0.9 kb K pnl fragm ent containing these sites has been sequenced. C om parative sequence analysis has dem onstrated that an elem ent w ith high hom ology to the consensus interferon-stim ulated response elem ent (ISRE) and the m urine MX gene ISRE sequence m aps to the m ost proxim al y-IFN induced b an d in the HS+8.7 doublet (Figure 5. 9. A and C). This proxim al site is rapidly induced during early Ly-6E.l y-IFN induced stages (Chapter 4. 2. 4.)

an d suggests the b in d in g of y-IFN in duced factors to enhance Ly-6E.l

expression. As several factors have the potential to bind to ISREs (review: Faisst an d M eyer, 1992) fo o tp rin t, gel shift an d functional assays are required to elucidate the specific factors binding to this element.

In addition to the ISRE elem ent at HS+8.7, potential factor binding sites w ere found for AP-3 and C /EB P corresponding to the location of the distal doublet at HS+8.7 (Figure 5. 9. A). Putative sites for PEA-1 and AP-1 w ere show n to correspond to the location of HS+8.9 (Figure 5. 9. B). Gel shift and functional analysis w ould determ ine w hether these transcription factors bind the identified consensus sequences at these sites and w hether these factors are required for the enhancing activity of this fragment.

5. 2. 5. In vivo analysis of the 3’ Ly-6E .l deletion constructs LGH-7 and

LGH-18

In o rd e r to d e te rm in e w h e th e r the d o w n s tre a m D N ase I h y p ersen sitiv e sites w ere required for in vivo expression an d w h eth er these sites delineated tissue specific regulatory elem ents, transgenic mouse analyses of 3' deletion constructs LGH-7 and LGH-18 were perform ed. As the hG H rep o rter has p rev io u sly been u sed successfully in transgenic expression analysis (M orello et aL, 1986; Pinkert et aL, 1987; Hanson et aL,

1991; D rezden et aL, 1992) and since the Ly-6E.l-hG H constructs were m ade

and tested in transfection experiments, constructs LGH-7 and LGH-18 were m icroinjected to produce founder transgenic mice. These constructs were chosen to determ ine the in vivo effects of Ly-6E.l 3' sequences from +2.94

Figure 5t IQ;

LGH-7 and LGH-18 transgene expression in the peripheral blood of founder mice.

A nalysis of peripheral blood RNA for transgene m essage w as perform ed by RT-PCR for Ly-6E .l-hG H fusion transcript and control

HPRT, on tail blood RNA.

(A). Quantification by FACS analysis of surface expression of the Ly-6A.2/Ly-6E.l antigens on peripheral blood leukocytes from a CBA (Ly-6E .l) X C57/B110 (Ly-6A.2) FI m ouse. M onoclonal antibodies

u se d are as follow s: D7, anti-L y-6A .2/ Ly-6E. 1 FITC lab elled

m onoclonal antibody; CD72, B cell specific PE labelled m onoclonal a n tib o d y ; CD4, T h elp er cell specific PE labelled m onoclonal antibody; CDS, T cytotoxic cell specific PE labelled m onoclonal antibody. The percentage of positive cells w ith in positive an d negative quadrants are shown.

(B). Ly-6E .l-hG H and control HPRT fragm ents generated by

RT-PCR of transgenic founder peripheral blood cDNA were detected by Southern blot to increase the sensitivity of the assay. Fragm ents w ere detected by hybridization w ith hG H and HPRT specific probes. Ly-6E.l-hG H transcripts are detectable in T2 (LGH-7) and T4 (LGH-18)

fo u n d er peripheral blood cDNA. The positive control cDNA used w as from u n in d u c e d NIH3T3 tran sfected w ith LGH-2, w hich e x p ressed hG H at 8 8 p g /m l/c o p y after 48 h rs (Figure 5. 5.).

Percentages depict the 1/10 dilution of initial 1 pg (10%), 0.1 pg (1%) and 0.01 pg (0.1%) control cDNA. The 225 bp Ly-6E .l-hG H fusion

tra n sc rip t an d 249 b p HPRT tran scrip t are distin g u ish ab le from genomic DNA contam inating bands at 500 bp and 1.1 kb respectively. M ark er frag m en ts from pUC18 restric te d w ith M spI is sh o w n alongside the blots.

A & 1 ô^‘ Tôi iè2 No Stain CD72 _{m -} j T i i n i i r — , ■ n n v . i j 1— r r T m n i— r r m - n

-fyO lAl lA2 lès lyH

TyO l y i lti2 i è s D7 D7 1 6 2 ' ï è s ’ "' i&w

B

#•

#

expression.

Three founders containing the LGH-7 transgene were produced and w ere found to contain ~1 (very low copy m osaic T l), 90-100 (T2) and 5-6 copies of the transgene by slot blot analysis. The male founder w ith 5-6 copies died suddenly at 6 weeks of age and thus expression analysis could

not be perform ed on this animal. Also, three founders w ere obtained from the LGH-18 microinjections, and contained 4 (T3), 100-110 (T4) and 2 copies of the transgene. The female transgenic with 2 copies died at 2 weeks of age and w as not analysed for transgene expression. A lthough transgenic mice w ith hG H reporter constructs have been reported to be larger than norm al (Pinkert et a l, 1987), none of the Ly-6E.l-hG H transgenics were significantly

larger than non-transgenic litterm ates.

Surface Ly-6E.l antigen is expressed highly on activated T and B

ly m p h o cy tes (K im ura et a l, 1984; Table 1. 2.). Therefore, to determ ine w hether the transgenes were expressed in peripheral T and B lymphocytic su b sets, RT-PCR analysis w as perform ed on fo u n d er p erip h eral blood RNA. N o rm al surface Ly-6E .l an d Ly-6A.2 an tig en ex p ressio n , as

determ ined by the D7 m onoclonal antibody staining and by FACS analysis, w as found on approxim ately 40% of peripheral blood leukocytes w ithin an CBA(Ly-6E .l) x C57/B110(Ly-6A.2) FI non-transgenic m ouse. D ouble

staining w ith D7 and lymphocyte specific antibodies dem onstrated that 25% an d 11% of D7 positive cells stained for T cell (CD4 and CD8) and B cell

(CD72) m arkers respectively (Figure 5. 10. A.). Figure 5. 10. B. show s the results of PCR Ly-6E .l-hG H and control HPRT p roducts generated from

p e rip h e ral blood cDNA. Both sets of prim ers w ere designed to span in tro n ic seq u en ces so as to d isc rim in ate b e tw ee n gen o m ic DN A contam ination and the presence of cDNA by fragm ent size determ ination. This analysis dem onstrated that LGH-7 founder T2 and LGH-18 founder T4 ex p ress the Ly-6E .l-h G H fusion tra n sc rip t w ith in p e rip h e ra l blood

leukocytes at 1% and 15% of control cDNA (calibrated w ith HPRT) from a p o p u latio n of NIH3T3 cells transfected w ith construct LGH-2 expressing hG H at 8 8 p g /m l/c o p y . No signal was found for the Tl or T3 founders.