Cloning procedures.

4.1 Generation of constructs»

CD2Nef constructs (see figure 13): The SOObp BamHl-Smal fragment from either pTG1147, pTG1191, pTG3191, pTG3132, or pTG2165 (gift from B. Guy) was blunted and ligated into a unique blunted EcoRl site in the first exon of the p2629 CD2 expression plasmid (gift from D. Kioussis) to give either p2629N47, p2629N91, p2629N391, p2629N32, or p2629N65. The orientation of the insert was tested using an Asp718/Hindin and a Xhol/BamHl digest (see map figure 13). A 4.5Kb BamHl-Notl fragment containing the 3 -CD2 LCR from p2694 (gift from D. Kioussis) was then ligated to the BamHl-Notl fragment from the p2629N... plasmids, resulting in either pCD2Nefl 147, pCD2Nefll91, pCD2Nef3191, pCD2Nef3132, or pCD2Nef2165. A Sall-Notl fragment was used to generate transgenic mice.

CD2Tat constructs (see figure 13): pCD2Tat72 was made as described for the CD2Nef constructs above. However, the original Tat fragment was excised using a Asp718/Sal 1 digest from plasmid pTZ19 (gift from Dr. E. Blair, Wellcome), which was then blunted into the EcoRl site of p2629. The orientation of the fragment was checked with a Hindlll digest (see map figure 13).

IL-2Nef constructs (see figure 14): The 789bp BamH 1-Sac 1 fragment from pTGl 147 was cloned into the BamHI-SacI sites of the bluescript vector. A 1.95kb Pstl-Hindlll fragment from the pATgMIL2-A plasmid (gift from Prof. W. Fiers - Uni. of Ghent, Belgium), containing the entire IL-2 promoter/enhancer and 44bp of untranslated 5* leader sequence, was then ligated into the Pstl-Hindlll fragment of the Nef/bluescript vector (this contained the bluescript backbone) to give pIL47. pIL47 was then cut with Hindlll, blunted and then cut with EcoRl to generate a 2.75kb blunt/EcoRI IL-2 promoter/enhancer/zi^gene fragment. This was ligated into the p2629 hCD2 expression cassette which had been prepared by first cutting with Sail, blunted and then cut with EcoRI. The resulting plasmid was pIL-2Nefl 147. An Apal-Notl fragment was used to generate transgenic mice.

4,2 Ligations.

In the cloning process, 10-50ng of linearised plasmid and lOOng of the insert fragment were mixed together in %n\ of HgO. To this, Ifil of lOx ligation buffer and l/il of T4 DNA ligase (lU//il) were added. The reaction was incubated overnight at 16**C. For transformation into E. coli 2/il of these samples were used.

4 3 Competent bacteria.

For transformation, CaClg competent E. Coli (strains DH5a or DHIO/S), were prepared. A single E. Coli colony was innoculated into a 20ml LB media culture and grown overnight at 37*’C with vigorous shaking. 100/a1 of this culture was then added to a fresh flask containing

250ml of pre-warmed LB media and incubated at 37®C. The OD«,o was monitored at regular intervals until it reached 0.5. The culture was then cooled rapidly in an ice bath, followed by the centrifugation of the bacteria at 4k rpm for 10 min at 4**C. The cells were then resuspended in 62.5ml of ice cold sterile O.IM MgClg, and re-pelleted by centrifugation at 4k rpm at 4"C for 10 min. The cell pellet was resuspended in 31.25ml ice cold sterile O.IM CaClg and left on ice for 20 min. After centrifugation at 4k rpm at 4'C for 10 min, the cells were resuspended in 26.5ml of ice cold O.IM CaClg and 3.5ml of glycerol. 200/d aliquots of this solution were then snap frozen in eppendorf tubes and stored at -70®C until required.

4.4 Bacterial transformations.

The DNA to be transformed was mixed with 100/d of rapidly thawed competent cells and left on ice for 30 min. The mixture was then transferred to a 4TC water bath for 90 seconds, then onto ice for 5 min. 1ml of LB media was then added and the culture allowed to incubate at 37"C for Ihr. The cells were pelleted by centrifugation for 10 sec and all the supernatant, with the exception of 50/tl, removed. The cells were resuspended in this volume, then spread onto LB plates containing 100/ig/ml ampicillin. After overnight incubation at 37"C, single colonies were picked and innoculated to 5ml LB/ampicillin (100/ig/ml) cultures for miniprep purification of their plasmid DNA.

4.5 Plasmid miniprep purification.

Single plasmid-containing E. Coli colonies were innoculated into 5ml of LB media with 100/ig/ml ampicillin. The cultures were incubated overnight at 37*C. 1.5ml of the culture was transferred to an eppendorf and centrifuged at 14k for 30 sec, the supernatant being discarded. 270/il of TEN buffer was added, with the pellet resuspended by vortexing. To this, 30/il of 10% SDS was added, vortexed, followed by ISO/il of 2M NaOAc pH 5.2. After mixing well Ix phenol/chloroform and Ix chloroform extractions were performed. To the final aqueous layer 0.9ml of 96% ethanol was then added, the sample placed on ice for 5 min, and centrifuged at 14k rpm for 10 min to pellet the DNA. Hie pellet was washed with 70% ethanol, air-dried and resuspended in 50/d of TE. RNAse treatment using l/il of lOmg/ml RNAse A was then carried out for 30 min at 37**C, followed by a phenol/chloroform extraction, ethanol precipitation and resuspension of the final DNA pellet in 50/tl of TE pH 7.4.

4.6 Plasmid maxi prep purification.

A single bacterial colony, grown up in 5ml cultures as described for miniprep purification, was used to innoculate a 1 litre culture of LB media with 100/tg/ml ampicillin. This was incubated overnight at 37”C. The culture was then centrifuged at 4k rpm in a 1 litre bottle for 20 min, the supernatant being discarded. The bacterial pellet was then resuspended in 40ml of Ix glucomix by vortexing. To this, 80ml of 0.2M NaOH/1% SDS was added, mixing gently, and left to stand for 5 min. 40ml of ice-cold 5M KOAc pH 4.8 was then added, again mixing gently and leaving to stand for 5 min. The mixture is then centrifuged at 4k rpm for 20 min, then poured through 8 layers of cheesecloth to remove the debris. To the remaining supernatant, 0.6

vol. of isopropanol is added, with gentle mixing to precipitate the DNA. The DNA is then pelleted by 20 min centrifugation at 4k rpm, washed with 70% ethanol, air-dried for 30 min, and resuspended in 5ml TE pH 8.0. The solution of DNA was then transferred to a pre-weighed Falcon 50ml tube, and adjusted to 9g with TE pH8.0. To this, 10.2g of CsCl and 1ml of 5mg/ml ethidium bromide was added. The solution was then transferred to Quick-seal™ centrifuge tubes and sealed with a heat clamp. The samples were then centrifuged for 24hr at 56,000 rpm in a 70.1 Ti Beckman rotor. The lower supercoiled band of plasmid DNA was removed using a syringe and needle. This was transferred to a 15ml Falcon tube, made up to 4ml with HgO, and then mixed with 8ml of 96% ethanol. The DNA was pelleted by centrifugation at 4k rpm for 10

min, followed by a wash in 70% ethanol and resuspension in SOO/tl of TE pH 8.0. To this, 10/a1

of 0.5M EDTA and 5/d of 10% SDS were added, followed by 2x phenol/chloroform extractions and a chloroform extraction. To the final aqueous phase, 50/il of 2M NaOAc pH 5.5 and 1ml of 96% ethanol were added to precipitate the DNA. After 5 min on dry ice, the sample was centrifuged for 10 min at 14k rpm. The DNA pellet was washed in 70% ethanol, and resuspended in 450/il TE pH 8.0, plus 50/d of IM NaCl. To this 2/il of lOmg/ml RNAse A was added, and incubated at 37**C for 30 min. Then followed 2x phenol/chloroform extractions, Ix chloroform extractions, the addition of 50/tl of 2M NaOAc pH 5.5 to the final aqueous phase, and the precipitation of the DNA by the addition of 1ml of 96% ethanol. After pelleting the DNA by centrifugation at 14k rpm for 10 min, a 70% ethanol wash was followed by the resuspension of the DNA in 500/d of TE pH 8.0. The plasmid DNA was then stored at -20*C until needed.