Componentes Principales del Vehículo Híbrido

A locus control region (LCR) has been identified within the 3' flanking region of the human CD2 (hCD2) gene (Greaves et al, 1989). This LCR contains a strong T cell

specific enhancer and additional regulatory elements which are responsible for

maintaining an active chromatin structure (Lake et al, 1990). A human CD2 minigene which contains the natural hCD2 gene with all but the first intron deleted, together with 5 kb of 5' promoter sequence and 5.5 kb of the 3' LCR can direct T cell specific expression of integrated TCR a and p cDNA, murine CDS a cDNA and other genes in transgenic mice (Mamalaki et al, 1993, Zal et al, 1994, Robey et al, 1991). Transgene expression is independent of integration site specific effects and is transgene copy number dependent (Greaves et al, 1989). Herein lies part of the problem with the initial hCD2 minigene cassette. In order to achieve detectable levels of expression more than ten copies of the transgene were frequently required.

Furthermore, to achieve expression levels equivalent to endogenous TCR gene expression it was often necessary to generate mice carrying 50-100 copies of the transgene (Zhumabekov et al, 1995).

The vector used in the present work is derived from the initial hCD2 cassette. It includes 5 kb of promoter sequence and 5.5 kb of the 3' LCR. In the new cassette transgene cDNA is inserted into the second exon of the hCD2 minigene, leaving the promoter and the first exon intact. The start codon and other potential start codons located upstream of the reporter gene (transgene) have been mutated and an artificial poly-linker containing EcoRl, SnaBl and Smal restriction sites has been engineered into the vector at the end of the first intron (Zhumabekov et al, 1995). All other hCD2 coding sequence has been removed and a 500bp fragment from the 3' untranslated region retained which contains two hCD2 poly-adenylation signals (Lang et al, 1988). The hCD2 minigene resides in the Bluescript SK(“) plasmid and the entire VA-hCD2 vector is 14.2 kb in size, figure 3.10.

Several TCR transgenic lines have been generated using this vector (Moskophidis and Kioussis, unpublished and Zhumabekov and Kioussis, unpublished) and transgenic mice carrying as few as 2-4 copies of the transgene ( a and p chains) been shown to

express transgenic TCR on the surface of peripheral T cells at levels similar to or higher than that of endogenous TCRs. This contrasts with the situation using the old hCD2 cassette in which no expression was detected in any of six transgenic lines carrying 5-15 copies of TCR a and p TCR cDNA (Moskophidis and Kioussis, unpublished and Zhumabekov and Kioussis, unpublished). The VA-hCD2 vector therefore represents a highly efficient mechanism for expressing transgenes in an integration site independent, copy number dependent T cell specific manner.

The VA-hCD2 expression vector has three neighbouring restriction sites into which insert DNA can be sub-cloned; EcoRl, SnaBl and Smal, figure 3.10. Digestion of the vector with EcoRl produces cohesive ends while digestion with SnaBl or Smal both produce blunt ended linearised vector. The cDNA library from clone 4A was initially constructed such that each cDNA molecule was flanked with BstX-l/EcoRl adapters. This enabled cDNA to be cloned into the library vector (pcDNAII) using the rare cutting enzyme BstX-1. pcDNAII has a multi-restriction enzyme poly-linker with the BstX-1 site towards the centre. Insert cDNA can therefore be retrieved from the library following digestion with either EcoRl, BstX-1 or any of a series of restriction enzymes which cut within the poly-linker either side of the BstX-1 site.

The TCRB V8 S3 transcript (p6) has two internal EcoRl sites: one within intron one and a second immediately 5' of the stop codon. It was initially hoped to clone TCR a and p cDNA into VA-hCD2 using the restriction enzyme EcoRl. However, digestion with EcoRl results in the production of multiple fragments, figures 3.8 B and 3.13 C. EcoRl is the only enzyme which generates cohesive ends and is suitable for use in sub-cloning into the VA-hCD2 vector. Since ligations involving cohesive ends are theoretically easier to achieve than those involving blunt ends a partial digest strategy was adopted to try and obtain a full length TCRB V8 S3 transcript. Unfortunately generation of the full length p6 transcript was very poor (even when the restriction

enzyme concentration was limiting) so much so that it was thought unlikely to be able to recover a sufficient amount of full length insert for successful sub-cloning.

As an alternative approach neighbouring restriction sites Xbal and BamHl (3' and 5' of the BstX-1 site) within the pcDNAII poly-linker were used to excise the (36 transcript. Both of these enzymes produce 5' overhangs which were subsequently blunt ended using the Klenow fragment of DNA polymerase I. The a 10 transcript was excised from the library vector pcDNAII using EcoRl. The a 10 and (36 transcripts were gel purified, sub-cloned into VA-hCD2 which had been linearised with either EcoRl or Smal respectively and transformed into Epicurian coli (Stratagene).

Twenty antibiotic resistant VA-hCD2-alO and twenty-eight VA-hCD2-(36 colonies were picked and plasmid DNA prepared. An EcoRl digest of VA-hCD2-alO and VA-hCD2-(36 DNA is shown in figure 3.11 A and B. Miniprep digest VA-hCD2-alO number 11 displays the correct digest pattern of large vector band (14.2 kb) and correct size insert band. The majority of the other VA-hCD2-alO miniprep digests seem to indicate that the VA-hCD2 vector has rearranged itself within the host

bacteria. Miniprep digests VA-hCD2- (36 numbers 2, 3,4, 19 and 20 all appear to have correct sized insert bands. The banding pattern of all other VA-hCD2- (36 miniprep digests once again suggests that rearrangement of the vector has occurred within the host bacteria (in the case of (36 ligation four attempts at ligation and transformation of bacteria were required and over 160 colonies screened before the correct digest pattern was observed). Since this work was completed other people in this laboratory have experienced similar problems with the VA-hCD2 vector.

Since the sub-cloning of p6 and alO cDNA into the VA-hCD2 vector is non- directional potentia^positive colonies must have the orientation of the ligated insert checked. This was achieved by using a discriminatory PCR method, figure 3.12. As shown in figure 3.13 A the TCRA V8 S13 transcript (a 10) has been ligated into the

VA-hCD2 vector in the correct orientation in VA-hCD2-alO colony ll(VA -hCD2- a lQ ll). Furthermore, the TCRB V8 S3 transcript (p6) has been ligated into the VA- hCD2 vector in the correct orientation in VA-hCD2 p6 colonies 2 and 20 and is present in VA-hCD2 p6 colonies 3, 4 and 19 in the wrong orientation, figure 3.13 B (I and II).

Colonies VA-hCD2-alO^^ and VA-hCD2-p6^9 were selected and plasmid DNA prepared. A check EcoRl digest was performed and revealed the correct banding patterns for both a and p transcripts, figure 3.13 C. Finally, the Bluescript SK(-) vector was excised and the a and P transgenes gel purified. Injection of (CBA x B6) F2 eggs was performed by Dr. Colin Hetherington at the Biomedical Services

Transgene Unit, John Radcliffe Hospital, Oxford. To date a single founder transgenic for both the a and p chain and a single founder transgenic for only the a chain have been obtained. Unfortunately, FACS analysis of thymocytes derived from transgene expressing offspring using TCR a and p chain specific monoclonal antibodies reveals no surface expression of either chain. Further injections are ongoing.

Figure 3.1: Examination of genomic rearrangement within the TCRB locus using a Southern blot technique: pictorial representation. (A) following digestion of genomic DNA with the restriction enzyme Hind HI and detection with the TCRBJ2 probe, (Palacios and Samaridis, 1991) a 5.2kb band is obtained from an unrearranged TCRB locus. (B) Rearrangement within the TCRB locus results in the apposition of specific V, D and J segments. This recombination results in novel digestion products the size of which will vary depending on the TCRB V segment which has undergone

recombination.

Figure 3.1

(A) Genomic rearrangement of the TCRB locus prior to VD.T recombination

T C R B V B D 2 BJ2 S I -6 B C 2

5’

BJ2 P r o b e

y

5:2 kb

Digestion with Hind III and Southern analysis using the BJ2 specific probe produces a 5.2kb band

(B) Genomic rearrangement of the TCRB locus post VD.T recombination

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