I don't think there is any problem using your packaging and envelop plasmids. I do it all the time and have no issue, although I am using Clontech's cherrypicker lentivirus. "4th generation" is just a term for advertisement, I think... 

I too am looking for some answers as to whether the Lenti-X expression vector is compatible with the 2nd or 3rd generation packaging system. 

The Clontech datasheet says that their 4th generation Lenti-X supplies 5 plasmids: 1. pTre-gag-pro, 2. LTRHIV-vpr-pol, 3. pCMV-VSVG, 4. pMV-tet-off, 5. pTre-tat-ires-rev

So tat is supplied on the 5th plasmid and should work with a 2nd generation lenti transfer vectors when pPAX2 + pMD2-G is supplied. Is this right?

With regards to compatibility with 3rd generation expression vectors, they mentioned: We have not tested all vectors, but in principle, any HIV-1-based vector should be efficiently packaged using this system, and higher titers compared to a 3rd-generation packaging system should be obtained. For example, when a ZsGreen1 cassette was cloned into the pLenti6/V5 vector (Invitrogen)—a vector that lacks the WPRE and cPPT/CTS sequence elements and utilizes self-inactivating (SIN) LTRs—titers of >3 x 107 IFU/ml were obtained as determined by flow cytometry. Since the Lenti-X system utilizes Tat transactivation, HIV-1 LTR-based vectors will generate higher titers than those containing other LTRs. Vectors lacking WPRE and/or cPPT/CTS sequences yield consistently lower titers than Clontech® pLVX vectors.

Invitrogen says The pLenti-DEST vectors contain the following elements:

• Rous Sarcoma Virus (RSV) enhancer/promoter for Tat-independent

production of viral mRNA in the producer cell line (Dull et al., 1998)

• Modified HIV-1 5′ and 3′ Long Terminal Repeats (LTR) for viral packaging and reverse transcription of the viral mRNA (Dull et al., 1998; Luciw, 1996)

Note: The U3 region of the 3′ LTR is deleted (∆U3) and facilitates self-inactivation of

the 5′ LTR after transduction to enhance the biosafety of the vector (Dull et al., 1998)

• HIV-1 psi (Ψ) packaging sequence for viral packaging (Luciw, 1996)

• HIV Rev response element (RRE) for Rev-dependent nuclear export of unspliced viral mRNA (Kjems et al., 1991; Malim et al., 1989)

Any comments appreciated!