Problemas en el Departamento de Crianza y Pesca

3.2.11.1. Preparation of scavenger receptor promoter gene

To determine the molecular mechanisms responsible for PMA and cytokine-dependent scavenger receptor transcriptional activation in HMC, the scavenger receptor promoter- luciferase fusion gene pGLSSCR was constructed. This contains scavenger receptor promoter 5’ upstream (-696 to + 46) from the scavenger receptor gene (Fig.3.3) (Wu et al. 1994). 5’upstream of scavenger receptor was a gift (Fxbal AI) from Dr. CK Glass.

A antisense primer was designed to sequence the upstream region o f scavenger receptor (solid bar). Sequence primer 5’CAGGGTGGAGTGCAGTGGTGTG 3’, which locates in scavenger receptor promoter region underlined (Fig.3.3).

3.2.11.2. Preparation of pGL3 enhancer vector

pGL3 enhancer vector was digested by Sad /Bgl II to produce sticky end for ligation as described in chapter 3

3.2.11.3. Ligation of pGL3 enhancer and scavenger receptor promoter

pGL3 enhancer vector digested by Sac I/Bgl II and scavenger receptor promoter with Sac I/Bgl n adapters was ligated using standard method described in chapter 2. The ratio of pGL3 enhancer and scavenger receptor promoter of ligation is 3/1. The ligation product was transformed to JM109 cells, and then subjected to selection. The positive clones were selected for mini-preparation, identification and maxi-preparation of plasmid using the methods as described in chapter!. The plasmid pGL3SCR was used for transfection experiments.

FxbaAI

-4kb -696 bp -490bp +46bp

î

BamHI Xbal Xbal Xbal HindIII

Sequence direction -907 -226 - 4 9 0 a t t a a t g t a t g t t t t a g a a g g c a t a g t t a c t t a t a a a a a a g g a a a g a t c a g g c t g g g c a c - 4 3 0 g g t t c g c g c c t g t a a t c c c a g c a c t t t g g g a g g c c a a g g c g a g c g g a c c a t g a g g t c a g g - 3 7 0 g g a t c a a g a c c a t c c t g a c c a a c a t g g c g a a a c c c t g t c t c t a c t a a a a t a c a a a a a a t t - 3 1 0 a g c c g g g t g t g a t g g c a c a c g c c t g t a g a a c c t g g g a g g c a g a g g t t g c a g t g a g c t g a g - 2 5 0 a t ca.ca.cca.c t g c a c t c c a c c c t g g t g a g a c a g c g a g a c t c c a t c t c c a a a a a a a a a g g a

^ 3'gtgtggtq acqtqaqqtq qgac 5 ' (sequsncing primer)

- I B O a a g c t c a a t c t g c t g t a a a t t a t g t g c t t g t t t c a a c a a c c c t t g t t t c t t t t c c t t t t c - 1 3 0 a c t t c t c t t t t t t t t t t a a a g c g g c c t a a a t g g g g t g a a g a g t g a g t t a t c t g a c a a a t t - 7 0 t a g a t t t t g c a a a c c t g t g c a t t g a t g a g a g t g c t a t t g a a a c a c a t t a a g a a a g a t t t t - 1 0 c a a c g c a g g a a t g t g t c a t t t c c t t t c t t c a t g t a c c a g a t g c t g a a a t a ctatgaqata

(PCR lower primer) 3 ' g t c t a c g a c t t t a t ç r a t a c t 5 '

+50 aagattttag gtttcaattg taaagagaga gaagtggata aatcagtgct gctttcttta +110 ggacgaaag

Fig.3.3. S tru c tu re a n d sequence of sc av e n g er re c e p to r p ro m o te r. T he sequence show n above represent the open b ar region. T ranscription start point A TG has show n in italic bold. T he sequence

in solid bar region is unclear.

The sequence (antisence) o f 5’upstream o f scavenger receptor gene was performed by MWG-biotech and sequence show following:

- 2 2 6 5 ' TGCAACCTCTGCCTCCCAGGTTCTACAGGCGTGTGCCATCACACCCGGCTAATTTTTTGTATTTTAGT AGAGACAGGGTTTCGCCATGTTGGTCAGGATGGTCTTGATCCCCTGACCTCATGGTCCGCTCGCCTTG GC CTCCCAAAGTgCTGGGATTACAGGCGCGAGCACCGTGCCCAGCCTGATCTTTCCTTTTTTATAAGTAA TCTATGCCTTCTAAAACATACATTAAT - 4 9 0 - 4 9 1 TCTCATGATCTCCTCTTTCACGTTACTATAATGTTGAGTAAAGACTCCATGTTTGGCTAGGTGTGGTG GCTCATGCCTGTAATCCCAGCACTTTGGGTGGCCGAGGTGGCTGGATSSACTTGAGGTCAGGAGCTCT AGAGCGGCCGCTCTAGAATGAGGTCCATGTAAGATTATGCCCAGTGACCAAACAGAGATT CATAATCAAATAGGCAAATGGCTTGAGAGAATTGATTCAAATGTCCTAGGACTCACTGTGGTACAGGT AT