15 replies to this topic

[Kami23]

Hey all,

what would you say is the biggest insert you could put into a viral vector and then transfect into a mouse?

[scolix]

Kami23, on Feb 6 2009, 05:04 PM, said:

Hey all,

what would you say is the biggest insert you could put into a viral vector and then transfect into a mouse?

Depends on the virus that you are planning to use. AAV has less than 5kb capacity and lentivirus around 8kb and adenovirus has nearly 15kb (I am not exactly sure) and there are others with even higher capacity.

[Kami23]

scolix, on Feb 6 2009, 04:38 PM, said:

Kami23, on Feb 6 2009, 05:04 PM, said:

Hey all,

what would you say is the biggest insert you could put into a viral vector and then transfect into a mouse?

Depends on the virus that you are planning to use. AAV has less than 5kb capacity and lentivirus around 8kb and adenovirus has nearly 15kb (I am not exactly sure) and there are others with even higher capacity.

thanks... we are looking at about a 10kb insert...

[cellcounter]

scolix, on Feb 6 2009, 09:38 AM, said:

Kami23, on Feb 6 2009, 05:04 PM, said:

Hey all,

what would you say is the biggest insert you could put into a viral vector and then transfect into a mouse?

Depends on the virus that you are planning to use. AAV has less than 5kb capacity and lentivirus around 8kb and adenovirus has nearly 15kb (I am not exactly sure) and there are others with even higher capacity.

I know that lentiviruses have more cargo capacity than retroviuses. Beyond a particular size, the viral titer that you get will start getting down drastically. Not sure of precise numbers on insert sizes.

[Kami23]

cellcounter, on Feb 6 2009, 04:49 PM, said:

scolix, on Feb 6 2009, 09:38 AM, said:

Kami23, on Feb 6 2009, 05:04 PM, said:

Hey all,

what would you say is the biggest insert you could put into a viral vector and then transfect into a mouse?

Depends on the virus that you are planning to use. AAV has less than 5kb capacity and lentivirus around 8kb and adenovirus has nearly 15kb (I am not exactly sure) and there are others with even higher capacity.

I know that lentiviruses have more cargo capacity than retroviuses. Beyond a particular size, the viral titer that you get will start getting down drastically. Not sure of precise numbers on insert sizes.

oh and could you use the cDNA instead of amplified DNA for the insert?

[cellcounter]

Kami23, on Feb 6 2009, 09:51 AM, said:

cellcounter, on Feb 6 2009, 04:49 PM, said:

scolix, on Feb 6 2009, 09:38 AM, said:

Kami23, on Feb 6 2009, 05:04 PM, said:

Hey all,

what would you say is the biggest insert you could put into a viral vector and then transfect into a mouse?

Depends on the virus that you are planning to use. AAV has less than 5kb capacity and lentivirus around 8kb and adenovirus has nearly 15kb (I am not exactly sure) and there are others with even higher capacity.

I know that lentiviruses have more cargo capacity than retroviuses. Beyond a particular size, the viral titer that you get will start getting down drastically. Not sure of precise numbers on insert sizes.

oh and could you use the cDNA instead of amplified DNA for the insert?

What do you mean? You can use any DNA, be it intron, exon, promoter, ORF, cDNA, complete gene and shRNA. Of course, the most common use is to clone complete cDNA sequence and get a gene expressed that way. But these days retro/lentiviral shRNA has become very common.

[Kami23]

I would be more useful for me to use the cDNA because we are having a problem amplifying the C-terminal of the gene in question (but i think that was a primer problem).  Also if it is possible to do it this way and express it in the eye alone, would it be a reliable way of testing our hypothesis? I mean would it be better to clone the whole gene or just stick with the cDNA which I know is error free?

[cellcounter]

Kami23, on Feb 6 2009, 10:02 AM, said:

cellcounter, on Feb 6 2009, 04:57 PM, said:

Kami23, on Feb 6 2009, 09:51 AM, said:

cellcounter, on Feb 6 2009, 04:49 PM, said:

scolix, on Feb 6 2009, 09:38 AM, said:

Kami23, on Feb 6 2009, 05:04 PM, said:

Hey all,

what would you say is the biggest insert you could put into a viral vector and then transfect into a mouse?

Depends on the virus that you are planning to use. AAV has less than 5kb capacity and lentivirus around 8kb and adenovirus has nearly 15kb (I am not exactly sure) and there are others with even higher capacity.

I know that lentiviruses have more cargo capacity than retroviuses. Beyond a particular size, the viral titer that you get will start getting down drastically. Not sure of precise numbers on insert sizes.

oh and could you use the cDNA instead of amplified DNA for the insert?

What do you mean? You can use any DNA, be it intron, exon, promoter, ORF, cDNA, complete gene and shRNA. Of course, the most common use is to clone complete cDNA sequence and get a gene expressed that way. But these days retro/lentiviral shRNA has become very common.

I would be more useful for me to use the cDNA because we are having a problem amplifying the C-terminal of the gene in question (but i think that was a primer problem).  Also if it is possible to do it this way and express it in the eye alone, would it be a reliable way of testing our hypothesis? I mean would it be better to clone the whole gene or just stick with the cDNA which I know is error free?

1. For expression, you only need cDNA.

2. For various levels of expression, sometimes you need to include some known introns (I think globin field uses an intron to get max expression of globin cDNA. You need to check literature.

3. For in-vivo analysis, you can make retro/lentiviruses and microinject them into precise cells into retina. I think Adenoviruses are great for pulmonary infection.

4. There are other ways of overexpressing genes in eye alone, like electroporation, making specific promoter transgenic etc, that do not require making a virus.

Edited by cellcounter, 06 February 2009 - 08:11 AM.

[Kami23]

thanks thats really helpful the only problem with number 4 is our gene doesnt exist in mouse (well it did, but has now lost all function).  Ill try to find a way to use these ideas in my project planning I will be back with more questions though

[scolix]

Kami23, on Feb 6 2009, 06:12 PM, said:

thanks thats really helpful the only problem with number 4 is our gene doesnt exist in mouse (well it did, but has now lost all function).  Ill try to find a way to use these ideas in my project planning I will be back with more questions though

AAV serotype 2 is good for retinal ganglion cells. But ofcourse has a limitation. There is one paper which cut a gene into 2 and cloned each fragment into 2 different AAV and injected the virus. They later detected the complete protein. Just one way of using existing vectors for experiments

[Kami23]

scolix, on Feb 6 2009, 05:22 PM, said:

Kami23, on Feb 6 2009, 06:12 PM, said:

thanks thats really helpful the only problem with number 4 is our gene doesnt exist in mouse (well it did, but has now lost all function).  Ill try to find a way to use these ideas in my project planning I will be back with more questions though

AAV serotype 2 is good for retinal ganglion cells. But ofcourse has a limitation. There is one paper which cut a gene into 2 and cloned each fragment into 2 different AAV and injected the virus. They later detected the complete protein. Just one way of using existing vectors for experiments

you dont happen to have the citation for that paper do you? im going to plan everything thoroughly this weekend so i can present my plan to my supervisor on monday.  I just thought of something but now i cant remeber.... ah well it will come back to me lol

[Kami23]

I remeber now... say I used the cDNA then would I have to get a vector containing the promotor or would i have to somehow make my own?

[scolix]

Kami23, on Feb 6 2009, 06:25 PM, said:

scolix, on Feb 6 2009, 05:22 PM, said:

Kami23, on Feb 6 2009, 06:12 PM, said:

thanks thats really helpful the only problem with number 4 is our gene doesnt exist in mouse (well it did, but has now lost all function).  Ill try to find a way to use these ideas in my project planning I will be back with more questions though

AAV serotype 2 is good for retinal ganglion cells. But ofcourse has a limitation. There is one paper which cut a gene into 2 and cloned each fragment into 2 different AAV and injected the virus. They later detected the complete protein. Just one way of using existing vectors for experiments

you dont happen to have the citation for that paper do you? im going to plan everything thoroughly this weekend so i can present my plan to my supervisor on monday.  I just thought of something but now i cant remeber.... ah well it will come back to me lol

I think its from a Xiao lab. Try searching for it in pubmed. I will try to search and if I find it will post it here.

[Gongfu Panda]

Kami23, on Feb 6 2009, 09:04 AM, said:

Hey all,

what would you say is the biggest insert you could put into a viral vector and then transfect into a mouse?

Did you get your answers? I am planning to subclone 7.5kb cDNA in retrovirus or lentivirus vector so that I can make stable cell pools.

[moleculardelivery]

This may be a little late, but there are several publications out there using IN VIVO and EX VIVO electroporation in the eye - retina and otherwise.  I am told it can be very selective spacially once you become proficient at the technique.