Reporter Gene Constructs

This section describes three different types of reporter gene plasmids. All constructs were sequenced (see section 4.2.2) across ligation junctions to confirm exact boundaries. The method for large scale CsCl preparative gradients is described in section 2.2.3.

3.2.3.1 CAT vectors

pSVOCAT and pRSVCAT are described in Gorman et al. [1982a and b]. pSVOCAT is shown in Figure 3.1.

SV40 polyadenylation site pSVOCAT 4504 bp SV40 small t intron 5-GCG GTATTTTCTCCTT-3' \ sequencing primer V \ RC44 , CAT Gene

Hin dill 3'-GGG TTA CCG TAG CAT-5’ cloning sequencing primer

site RC37

Figure 3.1 pSVOCAT. The diagram shows the Hindlll cloning site and position of sequencing primers RC44, which extends down toward the CAT gene, and RC37 which extends back from the CAT gene.

The first mIL-5CAT construct was generated by subcloning a PCR product. This was obtained using the 5' primer PB1 (which binds to the region -547 to -516, and has a Hindlll site on its 5' end) and the 3' primer PB2 (which binds to the region +45 to +74, and has an EcoRI site on its 5' end) on D10.G4.1

genomic DNA. This fragment was subcloned into M13mpl9 and its sequence determined. The M13mpl9 vector containing the 627 bp PCR product was digested with Hindlll (Promega) and Eco47III (Amersham). The Hindlll end was filled in using the Klenow fragment of DNA polymerase I (Promega). pSVOCAT was linearised at the Hindlll cloning site (located 37 bp 5' of the CAT gene coding sequence), the ends were filled in with Klenow fragment of DNA polymerase I and phosphatased using alkaline phosphatase (Boehringer- Mannheim). The 579 bp blunt-ended PCR fragment was ligated into the blunt- ended and phosphatased pSVOCAT vector. Constructs with the insert in both the forward and reverse orientation were prepared. The forward construct has 547 bp of mIL-5 5' of the CAP site and 25 bp of mIL-5 3’ of the CAP site.

The largest mIL5-CAT construct contained -3859 to +25 of the 5' mIL-5 genomic sequence relative to the CAP site. This construct (known as -3859mIL-5CAT) was created using a 3884 bp Hindlll to Eco47III fragment from pSV2neo-10kbmIL-5G (kindly provided by Dr H. D. Campbell) which had been prepared on a LMP agarose gel (section 2.2.7). The Hindlll site was filled in using the Klenow fragment of DNA polymerase I (Promega) and the fragment was cloned into the phosphatased and blunt-ended Hindlll cloning site of pSVOCAT.

Initial 5' truncation mutants were obtained by restriction digests of -547mIL-5CAT. The 5' end of this construct has an NheI site which was generated upon ligation of the two blunt Hindlll ends. Double digests were performed with Nhel and four other restriction enzymes unique to the -547 bp of mIL-5 sequence. The enzymes employed were Accl to create -458mIL-5CAT, Xbal to create -312mIL-5CAT, Nsil to create -140mIL-5CAT and EcoNI to create -88mIL-5CAT. Noncompatible sticky ends were made blunt using T4 DNA polymerase before the vector was purified on an LMP agarose gel (section 2.2.7) and religated before transformation into DH5a (section 2.2.1.1) for plasmid preparation (section 2.2.3).

3.2.3.2 Luciferase vectors

Luciferase vectors pSVOAL and pRSVL are described in detail in deWet etal [1987].

57 3.2.3.3 Whole gene reporter constructs

The HmdIII-Hiwdlll 10,040 bp mIL-5 genomic fragment was transferred from pSV2neo (kindly provided by Dr. H.D. Campbell) to pSP73 (Promega), a bacterial vector containing no eukaryotic promoter sequences. This construct was designated pSP73-10kbmIL-5G. A 10 bp linker containing a unique Mlul restriction site was cloned into the unique BsfEII site in the 3’ untranslated region of the mIL-5 gene, creating pSP73-10kbmIL-5G(M/wI). This minor insertion provided the basis for distinguishing between expression from the transfected mIL-5 construct and expression from the endogenous mIL-5 gene (Figure 3.2). An analogous construct, pSP73-10kbmIL-5G(EcoRV), contained an EcoRV linker at the same site.

Polyadenylation site 10 bp M/uI

BstEII linker

Exon IV was inserted

into

Exon III u n iq u e B stE ll

site

(see below).

Exon I

I

Exon II

Position of 10 bp M lu l linker in gene

5'-GTG ACA GTA CGC GTC-3'

3'-T CAT GCG CAG CAC TG-5' Unspliced mIL-5 RNA transcript

Intron I Intron II Intron III' ' *

I I I I I I I f I I

t

7

5'-ATT GAC CGC CAA AAA GAG AAG TG-3’ PCR primer PB20

3'-TCA TGC GCA GCA CTG GGA CTT-5'

Figure 3.2 mIL-5 whole gene-PCR reporter construct, showing how expression is detected by PCR primers PB19 and PB20.

A positive control for PCR analysis was constructed from a mammalian expression vector, pcEXV3 [Miller et al. 1985] carrying a 6457 bp mIL-5 genomic fragment (kindly provided by Dr H. D. Campbell). This fragment spans Xbal to H i n d ill and includes 312 bp of 5' mIL-5. pcEXV3 contains SV40 enhancer/prom oter sequences which generate high expression levels in

59 mammalian cells. This vector was modified to create a construct analogous to pSP73-10kbmIL-5G(EcoRV) by inserting an EcoRV linker into the unique BsfEII site of the mIL-5 gene. This positive control construct was designated pcEXV3- 6.5kbmIL-5G(£coRV). A second positive control pcEXV3-6.5kbmIL-5G(M/uI) was created by inserting an Mlul linker at the same site.

3.2.3.4 Polymerase chain reaction primers

The PCR approach to investigating mIL-5 gene expression required the construction of 6 different PCR primers (Figure 3.3).

PCR primers MF74 and MF75 were designed by Dr M. C. Fung to detect wild type mIL-5 expression. PCR primers PB19 and PB20 were designed to detect expression from the modified mIL-5 genomic construct carrying the Mlul linker. PCR primer PB26 was designed to amplify with PB19 in detecting expression from the modified mIL-5 genomic construct carrying the EcoRV linker. PCR primer PB21 was designed to test primers PB20 and PB26 in PCR reactions and also as a primer to verify the sequence across the Mlul and EcoRV linker inserts.

In experiments with the positive controls it was established that PB19/PB20 were far less effective PCR primers than PB19/PB26 (see Figure 3.10 B and C in Results).