VALORACIÓN DE RIESGOS

Initial identification of transgenic founders and transgenic mice in the subsequent generations was performed with PCR technique using the DNA from tail biopsies. This simple technique allows us to investigate a large number of animals in a very short time. However, transgene integration pattern can not be determined by PCR analysis. To this end, Southern blot analysis is necessary to determine the number of integration sites, the number of integrated copies, orientation of the copies within the integration site and the integrity of the construct.

Southern blot analysis showed that two bands of about 8 kb and 2.7 kb representing the endogenous Igfbp4 were detected in both transgenic and wild-type mice, and a transgene-specific band of about 5 kb, as large as the microinjection fragment, represented in all transgenic mice from both lines. Additional bands, a > 10 kb only present in the mice from line 1 and a 10 kb band only present in the mice from line 2, represent the genomic regions that flanked the transgene construct in its integration site, indicating different integration sites in the two transgenic lines. From the Southern blot, we can see that there were more copies integrated in the genome of line 2 than in line 1. However, the exact number of the integrated copies was not determined, as we rather focused on the determination of the abundance of expression of the transgene and its biological activity.

5.1.2

Transgene expression

The transgene H-2Kb-mcIGFBP-4 was found to be transcribed and correctly spliced in all tissues tested, including adrenal gland, bladder, brain, heart, kidney, liver, lung, skeletal muscle, ovary, salivary gland, skin, spleen, testis and thymus. However, elevated protein levels were only detected in the spleen, thymus, lung and kidney by [125I]-IGF-II ligand blot. The elevated protein in the spleen was confirmed to be IGFBP-4 by Western immunoblot using an antibody specific for IGFBP-4. The

biological activity of the transgenic IGFBP-4 was confirmed by its capability to bind recombinant human IGF-II in a Western ligand blot.

The expression pattern of the transgene was identical in the two different transgenic mouse lines, indicating that expression of the transgene H-2Kb-mcIGFBP-4 occurs independently of the integration sites. Except for spleen, thymus, lung and kidney, the IGFBP-4 protein levels were not elevated in the other tissues, suggesting that transgenic mRNA expression was high in the spleen, thymus, lung and kidney, and low in the others. This expression pattern was similar to the endogenous H-2Kb expression (Morello et al. 1986).

The H-2K gene is one of the class I genes of the major histocompatibility complex (MHC) of the mouse which encode the heavy chain of cell surface H-2 antigens. The MHC comprises a multigenic family including both classical H-2 (H-2K, H-2D and H-2L) and H-2 like (Qa-TL) genes (Weiss et al. 1984). Their expression is developmentally regulated: classical H-2 antigens are not expressed from the early embryonic stages of development, become active between embryonic day 11 and 13, and are expressed and present at the surface of all somatic cells (Morello et al. 1978). In the adult mouse, the endogenous H-2Kb mRNA expression is ubiquitous, although the levels are different among the tissues. Its expression is relatively high in lung, liver, spleen and lymph nodes, intermediate in thymus, kidney and heart, and very low in muscle, brain, pancreas and testicular germ cells (Morello et al. 1986). Cis-acting sequences required for establishing this developmental expression pattern are present in the H-2Kb gene itself, and distinct regulatory elements controlling the tissue specificity are restricted to the 2-kb upstream promoter sequence (Drezen et al. 1992).

The mouse H-2Kb promoter has been used by several investigators for generation of transgenic mice expressing hGH (Morello et al. 1986), c-myc (Morello et al. 1989), c- fos (Ruther et al. 1988), hIGF-II (Buul-Offers et al. 1995) and mutant forms of the TNFRp55 (Plitz et al. 1999). In these mice, a tissue specificity similar to the endogenous H-2Kb mRNA expression was observed. In the H-2Kb-mcIGFBP-4 transgenic mice reported here, transgene expression pattern resembled also the expression of the endogenous H-2Kb gene. Transgene expression was ubiquitous, being high in the spleen, thymus, lung and kidney, and low in the others. Unlike H-

2Kb-hGH and H-2Kb-hIGF-II transgenic mice, serum IGFBP-4 level was not increased in H-2Kb-mcIGFBP-4 transgenic mice, probably due to the low transgene expression in the liver. According to the property of the H-2Kb promoter as noted above, transgene expression under the control of this promoter should be high in the liver, as reported in the H-2Kb-hGH and H-2Kb-hIGF-II transgenic mice (Morello et al. 1986; Buul-Offers et al. 1995). This different expression in the liver is unknown. A possibility might be that the IGFBP-4 was degraded after its translation by protease in the liver. However, experimental evidence is needed to support this notion.

Comparison of transgene expression in both transgenic lines revealed that the expression in line 2 was higher than line 1. This result is positively correlated with the integration number of the transgene construct, suggesting a mechanism of gene expression regulated by gene dosage. Alternatively, this phenomenon could be due to a position effect.