about using the pLentiLOX 3.7 vector in the lentiviral system - (Oct/13/2006 )

Hi, Guys. Does anyone has the experience in using the pLentiloxp 3.7 vector to generate the shRNA in the lentiviral system? From my preliminary experiments, I know that this vector can be packed very well in the packaging system that I am using. However, I want to know the efficiency of the knocking down from this plasmid.

---

i think the knock down by shRNA vector is more related to its promoter and sequence than the viral vector. so if u have verified that the specific shRNA u have is knocking down efficiently then mostly it will knock it down using viral vector.

Ofcourse, this is assuming that u have good titres with the virus preps. If u have good titres, and u have verfied the shRNA sequence, then knockdown shouldnt b a problem.

whereas if u have low titres, then even if u have good shRNA sequence, u will not get a knockdown.

---

Thanks! For this specific vector, it has certain requirement for designning of the shRNA, for example, it has to start with a G (due to the requirement of the promoter). I know a target sequence works well for the gene that I am about to knock down. However, this target sequence doesn't start with a G. Can I just put a G at the 5' end of my target sequence? Does anyone has experience using this kind of target sequence with one nuleotide mistmatch at the end to knowck down gene without affacting the knocking down efficiency significantly?

---

is this G part of the promoter. It can b that its a part of the promoter , then u shouldnt have any problem.

---

Thanks! For this specific vector, it has certain requirement for designning of the shRNA, for example, it has to start with a G (due to the requirement of the promoter). I know a target sequence works well for the gene that I am about to knock down. However, this target sequence doesn't start with a G. Can I just put a G at the 5' end of my target sequence? Does anyone has experience using this kind of target sequence with one nuleotide mistmatch at the end to knowck down gene without affacting the knocking down efficiency significantly?

---

This G is not part of the promoter. I just don't know why it needs to start with a G at +1 position from the U6 promoter in this vector. If this G is not a part of this promoter, will it hurt the knocking down efficiency? Thanks

---

if the promoter requres it, then u have to insert the G . i dont think the people who made this vector overlooked it.

---