Refinement of lentiviral vector for improved RNA processing and reduced rates of self inactivation repair

BMC Biotechnology

Table 2 Marker rescue assay (n = 3)

Vector	IU recovered/IU added^a		Frequency of SIN 5' LTR^d	Frequency of complete 5' LTR^e
	Round 1^b	Round 2^c
pHIV-1SDmSneo	7.79 ± 0.62 × 10^-5	0.21 ± 0.09	0% (0/17)	100% (17/17)
pHIV1SDmSneoLTR	0.71 ± 0.12	0.22 ± 0.40	0% (0/15)	100% (15/15)
pRK1neo	1.14 ± 0.47 × 10^-4	0.11 ± 0.02	18% (4/22)	59% (13/22)
pRK1Δatt340/184neo	5.56 ± 1.33 × 10^-5	2.52 ± 1.73 × 10^-2	32% (4/11)	17% (2/11)

^aEach vector was used to transfect 293T cells and subsequently rescued by transfection with pCMVΔRnr and pHCMV-g. The virus rescued in round 1 was used in a second round of transfection/rescue. Mean ± SD. ^bThe titre of rescued virus as determined on A549 cells and normalised to the amount of virus initially added. ^cThe titre of the virus rescued in the second transfection and normalised to the amount of virus rescued in round 1. ^dIndividual neomycin resistant colonies from the second round titre assay were analyzed via PCR for the structure of the 5'LTR (see also Figure 8), pRK1Δatt340/184neo significantly different to pHIV-1SDmSneo (Fisher's exact Chi squared, p < 0.002). ^eRepair refers only to LTRs having a complete U3 sequence as determined from PCR analysis (pRK1Δatt340/184neo significantly different to pHIV1SDmSneo (p < 0.0001) and pRK1neo p < 0.05, Fisher's exact Chi squared). For pRK1neo 5 colonies produced PCR products of intermediate size and one colony out of 23 did not yield a PCR product. For pRK1Δatt340/184neo 5 colonies produced PCR products of intermediate size (see also Figure 9) and 8/20 colonies did not yield a PCR product. Colonies not producing PCR products were not scored in the results shown above.

ISSN: 1472-6750