DIAGNÓSTICO DEL SISTEMA DE LIMPIEZA DE LAS REJILLAS

The objective of this piece of work was to generate a number of HBV core and pre-core expression vector constructs. These HBV genes could then be expressed in a eukaryotic cell culture system, in order to generate target cells in HLA class I restricted cytotoxicity assays that expressed endogenously processed antigen. In order to do this constructs with two different promoters and two different antibiotic resistance markers were used, in order to give subsequent experiments flexibility, in terms of quantity of expression (as different promoters may have different degrees of activity in different cells) and also in the cell lines available for use (Gorman et al. 1982). The two cell lines used were L721.221 and CIR (Kavathas et al. 1980; Storkus et al. 1991). A subsequent experiment showed L721.221 was resistant to histidinol, but CIR was sensitive, necessitating the creation of a hybrid vector, with a RSV promoter and encoding for hygromycin resistance. Furthermore as it was intended to transfect the cell lines with two different genes (one expressing an HBV protein and one an HLA class 1 allele), a choice of selection markers allowed compatibility with a greater number of other expression vector constructs. This became apparent when a specific CIR cell line transfected with a mutated HLA-A68.1 allele was used. This line had already been transfected with a vector which coded for resistance to hygromycin (pHEBO), and so transfection of a second allele required an alternative selectable marker. So subsequently the pMEP4 and pS constructs were used in L721.221 and the pREP8 constructs in CIR.

These specific vectors were chosen because the cell lines to be transfected were Epstein- Barr virus (EBV) transformed lines and these vectors should replicate efficiently and be maintained at high copy number due to the presence of Epstein-Barr nuclear antigen 1 (EBNAl) binding sites (Chittenden et al. 1989). This would theoretically allow higher protein expression, than if an integrating vector was used, the copy number of the latter depending on the number of DNA molecules initially electroporated into the cell and then the number of copies integrating into the host genomic DNA. Balanced against this was the fact that as the vector does not integrate into the cell line, cells would have to be grown in selection continuously in order to avoid loss of the vector. As one of the hypotheses to be tested was whether cytotoxic T lymphocyte (CTL) responses could be

elicited in patients with chronic hepatitis B infection, and that these cells were likely to be at low precursor frequencies, then it was elected to attempt optimal antigen expression by using the EBV based vectors from Invitrogen® (Leek, Holland).

It was hoped to model the role of HBeAg in the CTL response, using expression constructs containing the different DNA molecules i.e. to study the relative roles of the cytotoxic responses to the pre-core/core and the core products (Liang et al. 1991; Carman et al. 1991b; Okamoto et al. 1990). Although the pre-core/core product possesses an amino terminal peptide that signals it for export via the endoplasmic reticulum, it may be that following synthesis it could still be processed via the HLA class I pathway and so peptides derived from the pre-core region of the genome presented to CTL (Schlicht and Schaller, 1989). Therefore, both genes were to be used in the transfection experiments. The general experimental strategy used to generate these constructs was according to standard molecular biological techniques (Sanbrook et al. 1989). However, during the course o f these experiments difficulties were encountered predominantly at the ligation step. Other stages of the procedure were relatively straight forward. Several factors control ligation efficiency. For a ligation to be performed efficiently the appropriate concentrations of vector and insert must be present. The concentrations used were according to predetermined guidelines based on the relative sizes of the vector. These have been mathematically quantified according to the concentration of free vector and insert ends available to interact and so during the course of these experiments optimal concentrations of DNA could be maintained. The enzyme used for the ligation reactions was T4 DNA ligase. This is a 68kd enzyme encoded by the T4 bacteriophage, which catalyses the formation of a phosphodiester bond between the 5'-phosphate and 3'- hydroxyl ends of two double stranded DNA termini. The reaction requires energy in the form of ATP, as in the initial step a ligase-AMP intermediate is formed, and the AMP subsequently transferred to the 5' DNA strand prior to formation of the phosophodiester bond with the 3' DNA terminus. This reaction proceeds more efficiently if the ligase is repairing a ‘nick’ in the DNA as would be the case in a cohesive ended ligation, however is less efficient if the termini are not stabilised by the formation of interchain hydrogen

when cloning using blunt ended ligations more colonies were screened in order to find a positive one. The efficiency of transformation of bacteria is a further factor in molecular cloning. The bacteria used were well characterised strains and were made competent using an accepted protocol. The use of keeping bacteria at low temperatures in an environment of calcium ions with a subsequent heat shock to 42°C has been shown to produce transformation efficiencies of up to 10* transformants per pg DNA (Liu and Rashidbaigi, 1990). It may be that the two strains of bacteria used in the protocol had different transformation efficiencies, however the efficiency of transformation was not formally tested during the current experiments. The problem of the low efficiency of cloning of the pre-core/core fragment into pREP8 was circumvented using a more efficient colony screening technique rather than increasing the efficiency of the reaction.

The end result of the work of this chapter was therefore to clone the desired genes, both core and pre-core/core, into the vectors pMEP4, pREP8 and pS so generate a number of constructs suitable for the subsequent experiments.

CHAPTER 3

TRANSFECTION AND EXPRESSION