The parental virus in which 301B/1 expressing monomeric red fluorescent protein (mRFP) fused to pUL47 (pUL47mRFP) has been previously described [54 (link)]. The invariant lysine (K157) of 301B/1 was mutated to an alanine (A) to generate a CHPK mutant clone (rCHPKmut) using two-step Red-mediated mutagenesis in GS1783 Escherichia coli cells. Briefly, the I-SceI-aphAI cassette from pEP-KanSII was amplified by PCR using Thermo Scientific Phusion Flash High-Fidelity PCR Master Mix (Thermo Fisher Scientific, Waltham, MA, USA) using the primers listed in Table 1 . Subsequently, the K to A mutation was repaired back to K157 using the same approach. The primers used are listed in Table 1 . Restriction fragment length polymorphism (RFLP) analysis, analytical PCR, and DNA sequencing using the primers listed in Table 2 confirmed all clones were correct.
r301Bs were reconstituted by transfecting DF-1-Cre cells with purified BAC DNA plus jetOPTIMUS® (Polyplus, New York City, NY, USA) using the manufacturers’ instructions. After 2–3 days, transfected DF-1-Cre cells were mixed and seeded with fresh primary CECs until plaques formed, then further propagated in CECs until virus stocks could be stored. All r301Bs were used at ≤6 passages for cell culture and animal studies.
r301Bs were reconstituted by transfecting DF-1-Cre cells with purified BAC DNA plus jetOPTIMUS® (Polyplus, New York City, NY, USA) using the manufacturers’ instructions. After 2–3 days, transfected DF-1-Cre cells were mixed and seeded with fresh primary CECs until plaques formed, then further propagated in CECs until virus stocks could be stored. All r301Bs were used at ≤6 passages for cell culture and animal studies.
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