Shuttle Mutagenesis of CMV Genomic Fragments

1. E. coli strain XZ95: B224/RDP146 (F- recA1[Alac-pro] rps, spectinomycinr) with plasmid, pOX38::m-Tn3/gpt (pOX38 carrying the Tn3 transposon containing tetra-cycliner and guanosine phophoribosyl transferase).

2. E. coli strain 70: NG135 (K12 recA56 gal-AS165 strA, streptomycinr) with plasmid, pNG54 (pACYC184 with tnpR, choramphenicolr).

3. Chemically competent E. coli strain DH5a: endAl hsdR17 supE44 thi-1 recAl gyrA relA1 A(lacZYA-argF) U169 deoR (®80 dlacA([lacZ]M15).

4. Tetracycline (Sigma, St. Louis, MO): 15 mg/mL in H2O; use at 15 pg/mL.

5. Chloramphenicol (Sigma): 30 mg/mL in ethanol; use at 30 pg/mL.

6. Spectinomycin (Sigma): 100 mg/mL in H2O; use at 100 pg/mL.

7. Kanamycin (Sigma): 25 mg/mL in H2O; use at 25 pg/mL.

8. Streptomycin (Sigma): 100 mg/mL in H2O, use at 100 pg/mL.

9. LB broth and LB agar plates.

10. QIAprep Miniprep kit (Qiagen, Valencia, CA).

11. Sequencing primer: FL110PRIM (5'-GCAGGATCCTATCCATATGAC-3').

Fig. 1. Elements of the shuttle mutagenesis procedure. (A) Schematic representation of the modified Tn3 transposon. It contains six elements: a loxP sequence (loxP), a tetracycline resistance marker (tet), guanosine phosphoribosyltransferase (gpt), an additional poly(A) signal, and two 38-bp terminal inverted repeats (IR). (B) Overview of shuttle mutagenesis. I, MCMV DNA that has been partially digested with Sail is cloned into the Sail site of pHSS6-SaiI and introduced into B211 containing pLB101, which expresses the transposase gene, tnpA. II, Dual plasmid bacteria are selected by resistance to Cm and Kan and are conjugated to XZ95 containing pOX38::m-Tn3/gpt to generate a triply resistant bacteria (Tet, Kan, and Cm). III, TnpA catalyzes the insertion of pOX38 into pHSS6 to form a co-integrated plasmid. pLB101 is immune to this process as it carries a 38bp IR. IV, The co-integrate is resolved by conjugation with a bacteria that expresses X(cre+) resolvase, resulting in a pHSS6-MCMV plasmid that now contains a single Tn3 insertion. V, The mutated plasmids are isolated and reintroduced into DH5a bacteria and selected for their double resistance to Tet and Kan. Colonies are screened for the presence of a genomic fragment that also contains a Tn3 insertion. The mutated fragments are sequenced at the Tn-MCMV DNA junction to determine the site of insertion.

2.3. Recombinant Virus Generation

1. NotI restriction enzyme (New England Biolabs).

2. Buffer-saturated 25:24:1 phenol/chloroform/isoamyl alcohol (Fisher Scientific).

3. n24:1 Chloroform/isoamyl alcohol (Fisher Scientific).

4. 3 M Sodium acetate (Fisher Scientific).

6. Full-length, wild-type murine CMV genomic DNA (Smith Strain, ATCC) from purified virions.

7. Murine NIH/3T3 fibroblasts (ATCC).

8. NIH/3T3 media (Dulbecco's modified Eagle's medium [DMEM] with high glucose, penicillin/streptomycin, nonessential amino acids, essential amino acids [Gibco/Invitrogen, Carlsbad, CA], 10% NuSerum [Becton Dickinson, San Jose, CA]).

9. Calcium phosphate transfection kit (Gibco/Invitrogen).

10. 25 mg/mL Mycophenolic acid in H2O; use at 25 |g/mL, (Gibco/Invitrogen).

11. 10 mg/mL Xanthine in 1 M NaOH; use at 50 |g/mL (Gibco/Invitrogen).

12. Type VII low Gelling agarose, tissue culture grade: 2% w/v in tissue culture grade water (Sigma).

13. 2X NIH/3T3 media (DMEM from powder; Sigma).

14. 10% Nonfat dry milk (NFDM) in tissue culture grade water, autoclaved (Gibco/Invitrogen).

2.4. Recombinant Rescue Virus Generation

The materials needed in this section are identical to those of Subheading 2.3. with the addition of:

1. Full-length, mutant murine CMV genomic DNA from purified virions.

2. Murine STO fibroblasts (ATCC).

3. STO media: DMEM with high glucose, penicillin/streptomycin, 10% fetal bovine serum (Gibco/Invitrogen).

4. 25 mg/mL 6-Thioguanosine in 0.2 M NaOH; use at 25 pg/mL (Sigma).

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