Expression of IGFBPl and IGFBPS in first and second trimester tissues

5.3.1 N orthern analysis

The expression of IGFBPl was initially examined in total RNA isolated from mid­ trimester fetal liver using Northern analysis. RNA samples extracted from placenta and chorion decidua collected at term were used as positive controls. These tissues were selected since high levels of expression of IGFBPl and IGFBP3 had previously been observed in maternal chorion decidua and placenta, respectively (Pannier et al., 1994). In order to determine the quantity of fetal liver RNA needed to detect expression, increasing amounts of RNA, ranging from 10 to 50 jig, were loaded on the gel.

Expression of IGFBPl and IGFBP3 was analysed by hybridising filters with probes for both genes simultaneously. Transcripts of the expected sizes were seen for both

IGFBPl and 3 (Figure 5.1). As expected, IGFBPl was expressed in chorion decidua but not placenta whereas IGFBP3 was expressed in both. IGFBPl expression in fetal liver was first detectable at a concentration of 30 |ig per lane and IGFBP3 expression at 20 |Xg. The IBS and 28S ribosomal RNA bands were also visible due to non-specific cross hybridisation with the probes. Although the IGFBPl and IBS ribosomal bands were close

Chapter 5 IGFBPl and IGFBP3

in size (approximately 1.8 kb), signals from the two transcripts could be readily differentiated.

In their study of IGFBP expression using Northern analysis, Pannier et al (1994) loaded 20 pg RNA per lane from a number of different fetal tissues (lung, heart, spleen, placenta, liver, kidney and brain). At this concentration, only low levels of IGFBPl

expression were seen in two out of three mid-gestation fetal liver samples. Since fetal liver is an abundant source of IGFBPl, it was predicted that a minimum of 20-30 pg RNA would be required to study expression in other fetal tissues using this technique. With RT- PCR, on the other hand, 1 pg total RNA can be used to generate enough cDNA template for 20 PCR reactions. Given the limited availability of fetal samples, this alternative method was used to analyse expression in the remaining fetal tissues.

Fetal liver 28S 18S IGFBPl 2.4 kb .8 kb

Figure 5.1. Northern analysis of IGFBPl and IGFBPS expression in mid-trimester fetal liver. Lane 1: term placenta (TP); lane 2: term chorion decidua (TCD); lanes 3, 4, 5 and 6: increasing concentrations of fetal liver RNA. Position of IGFBPl and IGFBPS transcripts and IBS and 28S ribosomal RNA bands marked on left; transcript sizes indicated on right.

5.3.2 R T-PCR

Total RNA was extracted from normal human fetal tissues from both first and second trimester. Three sets of tissues (placenta, skin, brain, heart, liver, heart, intestine, kidney and lung) from both 10-12 and 16-18 week stages were analysed. Each set of tissues was derived from several different fetuses. RNA was reverse transcribed and the resultant cDNA amplified using PCR. Primers for the housekeeping gene GAPDH were used to control for the presence of cDNA. These primers gave a 598 bp product in all tissues investigated (Figures 5.2A and B). Control samples prepared without M-MLV reverse transcriptase (RT negative) were used to confirm the absence of contaminating genomic DNA. Primers for IGFBPl and IGFBPS were designed to enable expression of both genes to be studied. As described in Section 5.1 above, the family of IGFBPs share structural similarity, particularly in their -NH^ and -COOH terminal regions. Primers for

IGFBPl and IGFBP3 were therefore designed to span the regions of lowest homology in exon 2 of both genes.

5.3.2.1 I G F B P l

Primers IGFBP 1-F and IGFBP 1-R were used to detect expression of IGFBPl.

Sequence analysis of the resultant 315 bp PCR product confirmed that it matched the published sequence for IGFBPl in the GenBank database (from nucleotides 3304 in exon 2 to 5663 in exon 4, accession no. M59316). The product spanned introns 2 and 3, allowing contamination of cDNA with genomic DNA to be easily distinguished. PCR using genomic DNA as template gave a faint band of 2420 bp in length, corresponding to the size predicted from the published sequence.

Using primers IGFBPl-F and -R, PCR was carried out at 35 cycles. The only fetal tissue found to consistently express IGFBPl at both gestations was liver. PCR products of the expected size (315 bp) were seen in all liver samples analysed from both 10-12 and lb- 18 weeks gestation (Figures 5.3A and B). Since the PCR cycle number used was outside the linear range, no conclusions could be drawn about the relative level of expression at first and second trimester stages.

Chapters IGFBPl and IGFBP 3 B 600- 500- 400" 10-12/40 16-18/40

F ig u re 5.2. Gel electrophoresis of RT-PCR products showing GAPDH expression in fetal tissues. Expression investigated at (A) 10-12 week gestation and (B) 16-18 week gestation. Tissue types are: placenta (P), liver (L), brain (Br), lung (Lg), gut (G), heart (H), skin (S) and kidney (K). M: 100 bp marker; Bl: blank (no template). RT negative (-) control samples confirm the absence of genomic contamination.

bp M

B

400—

10-12/40

16-18/40

F ig u re 5.3. Gel electrophoresis of RT-PCR products showing IGFBPl expression in fetal tissues. Expression investigated at (A) 10-12 week gestation and (B) 16-18 week gestation. Tissue types are: placenta (P), liver (L), brain (Br), lung (Lg), gut (G), heart (H), skin (S) and kidney (K). M: 100 bp marker; Bl: blank (no template).

Expression was also observed in other samples. For example, in Figures 5.3A and B, expression was seen in placenta and gut at both gestational ages. The band in placenta probably reflects contamination with maternal decidua, which is a rich source of IGBPBI,

since expression could not be demonstrated in other placental samples studied. Expression was seen in four out of the six fetal gut samples analysed. Various explanations for this observation were considered. Contamination with maternal decidua or fetal liver seemed unlikely since both are usually easily distinguished from intestine at collection. It was therefore postulated that at early gestations all foregut-derived structures (including liver, stomach and duodenum proximal to the bile duct opening) might express IGFBPL No distinction was made between large and small intestine when initial gut samples were collected. In order to test this theory, therefore, additional samples of stomach (foregut) and large intestine (mid/hind gut) were obtained from first and second trimester fetuses (data not shown). IGFBPl expression was absent in tissues from both sources. It is possible that there is biological variability between samples and regional differences in mRNA expression in the tissues from which RNA was extracted. Alternatively, expression may be due to contamination with tissues highly expressing IGFBPl such as maternal decidua or fetal liver.

The liver-specific expression pattern observed is consistent with previous reports of

IGFBPl expression in fetal tissues (Pannier et a l, 1994; Han et al., 1996). Subsequent investigation of the imprinting status of IGFBPl was carried out using liver samples alone.