I am trying to clone transcription factors such as sox2, klf4 and oct 4 and c-Myc using lentivirus and retrovirus in the fibroblast culture. I am very new in this field. can you guide me from where I should start it? I am naive in the cloning work....
Hi Ram, thanks for your comment! First, determine what promoter would be best. For example, if you're planning on using your lentivirus/retrovirus for iPSC reprogramming, a good choice could be EF1a or PGF promoter. Once you know what promoter you'd like to use, you can go ahead and find a lentiviral/retroviral vector that includes the promoter you chose followed by a multiple cloning site (like our lentiviral cloning vector: goo.gl/bbGPFC). Next, find and purchase the gene(s) you'd like to express (abm has a huge selection of genes in our ORF library: goo.gl/3PFduR). Next, design primers to amplify each gene insert. You may want to design them with a ~15 bp overhang homologous to the vector arm and followed by a restriction enzyme site, then the regular binding portion. After amplification, you should have fragments that look like: __vector arm homology__restriction site 1__GENE__restriction site 2__vector arm homology__ Next, you need to ligate your inserts to your lentiviral/retroviral vector. You'll first need to digest your vector using the two restriction enzymes. Follow this with seamless cloning (you can try our Pro Ligation-free cloning kit: goo.gl/2SHXVc) to ligate the fragments together. You'll need to do this for all the genes. Finally, you'll need to package the resulting vector(s) into your lentivirus/retrovirus and then infect your fibroblast culture. That's the gist of it in a nutshell! There are easier routes, of course - you could just buy a premade iPSC lentivirus (we have lentiviruses available here: goo.gl/wCLSVm as well as retroviruses available here: goo.gl/e6Wgs1) that's ready to infect your cultures. Since you're working with sox2, klf4, oct4, and c-Myc, it may interest you to know we have all 4 of these genes available as pre-packaged lentiviruses already - either individually or as a set: goo.gl/HG9SEB Cheers!
@@abmgood This was a great explanation. I didn't quite understand the first-part about choosing the promoter. The link didn't work for me. I have a few questions. What if I want my transgene to be expressed only by specific cells? If there is a gene that I have identified let's say Gene X, where protein X is expressed by my desired group of cells (Alpha-Cells), how do I determine the right promoter to make sure I can express my transgene (gene Y) in Alpha-Cells only? If you target embryonic cells or stem cells, how do you ensure only a subset of its differentiated progeny express the transgene?
Hi @@Matiasporrascool ! This is a challenging question to answer as it would depend on the cell type and whether you want expression ONLY in those cells and nothing else. 1) Controlling by delivery method: For example, different serotypes (determined by the capsid of the virus) of AAV can infect different tissue types. Eg. this table on our AAV page(www.abmgood.com/AAV-Adeno-Associated-Virus.html) 2) Using tissue-specific promoters, or genetic elements controlling expression levels or localization: Specifically for alpha-cells, the best option would be to comb through the literature to see if there are any proven promoters that work specifically with alpha-cells. Some resources that might be helpful are: academic.oup.com/mend/article/30/2/248/2556546 link.springer.com/article/10.1007/s00424-016-1864-z
Hi, 4:28 "self-inactivation system is created as the deletion is transferred to the 5' LTR after reverse transcription and integration" Could you give more detail regarding how the deletion can be transferred to 5' LTR?
Thanks for your comment, J Liu! In the life cycle of retroviruses, the U3 region of the 3' LTR is duplicated to form the corresponding region of the 5' LTR during the process of reverse transcription and viral DNA synthesis in infected cells. 133 bp in the U3 region of the 3' LTR, which contains the TATA box and binding sites for transcription factors Sp1 and NF-kB, is deleted and this gets transferred to the 5' LTR after reverse transcription. Consequently, the transcriptional unit from the LTRs in a provirus is eliminated. If you would like to know even more, here is reference ID showcasing the development of a self-inactivating lentivirus vector - PMID: 9733856. Hope this helps!
Hello. Would like to confirm if these recombinant lentivirus can infect an already infected CD4 Tcell especially those with reduced CD4 expression due to HIV accessory proteins.
Hi Ro, we use HEK 293T cells due to their ease of transfection, reliable growth rate, passaging ratios, and optimal size of cells. Furthermore, 293T cells have the "T" antigen (Hence 'T' addition from their parental HEK 293 cell line), which is crucial for lentivirus production to high copy number using plasmids that have an SV40 origin of replication (most lentiviral plasmids have this). Hope this helps!
Impressive so its viral replication is controlled by removing rna transcription factors and removing the dimer making further synthesis impossible. Then you're adding a broad range receptor surface for enhanced binding. But how is exact delivery determined then ? Wouldn't there be a possibility of nullifying or cross linking proteins that have ambiguity in binding receptor surfaces ? I just imagined the capsid surface would have a designer site specific receptor like one from an antigen or antibody.
Hello Fireteam Omega! Thanks for watching and leaving a comment! VSV-G has been shown to have broad cell tropism and lead to stable pseudotypes, making it a good choice for a wide range of applications. It's much simpler to use an already-existing coat protein with proven effectiveness in mediating viral entry than to custom-design a new coat protein. What determines the broader delivery is not that VSV-G binds to a greater variety of receptors than the env protein, but that VSV-G binds to one particular receptor family (the LDLR family), which is expressed widely in many cell types.
Either virus can be used to deliver sgRNA and Cas9, however, AAV's small packaging capacity means that it can only fit saCas9, not the more commonly used spCas9. AAV is preferred by some scientists due to its low immunogenicity and transient expression, especially for in vivo experiments. Lentivirus integrates to cause long-term expression, which can be useful for maximizing the possibility of success of the CRISPR experiment. However, some scientists worry that long-term expression of Cas9 might have unknown effects on their cell line. Hope this helps :)
Hi Lina, the main concern when expressing multiple genes with lentivirus is packaging capacity. If you wish to express both genes from one vector, the total length of the sequence between the LTRs shouldn't exceed 9 kb. If your cassette would be larger than this, you may wish to package each gene separately. Another decision is whether you want to express the genes from the same promoter, but separated by an IRES or 2A peptide, or from two separate promoters.
Hi Suneetha, thank you for reaching out to us and we'd be happy to help you! Would you like to send your question to our experts in our technical team? You can email them directly: technical(AT)abmgood(DOT)com
Will Lentivirus spread to infect more cells? How effective is it to get to target tissue to have an effect with a single injection? Is does it need multiple injections?
Hi Justin, thanks for leaving your comment! All of our lentiviruses are replication incompetent - meaning they cannot replicate on their own to infect more cells once the virus is packaged. Single injection is highly effective, as Lentivirus has a relatively high transduction efficiency, and is stably integrating. Usually an MOI of 5-20 will work for any non-hard to transduce cell lines. Hope this helps!
@@abmgood yes thanks for the reply! Can lentivirus be used for multiple therapies? I know a built up immunity to AAV can be a problem. I'm wondering if there is the same problem with lentivirus.
@@JC-ly8pz Yes, it seems lentivirus can be used for multiple therapies, as immune responses in murine studies (either already existing, or acquired from multiple administrations) do not reduce the efficacy of lentiviral vectors! If you would like to read more, I would recommend this Paper in BMJ: thorax.bmj.com/content/72/2/137
Hi Shayomi - thanks for leaving a comment! The process described is for applications of recombinant HIV Lentivirus systems in cell biology. That being said, there are 3rd generation systems for using SIV for gene delivery as well (though they are less commonly used) that are based on similar principles of safety and robust gene delivery. See this article on the use of SIV in Cystic Fibrosis gene therapie: goo.gl/VfD3ru
Post your questions here! And learn more about the Recombinant Lentivirus System at our knowledge base: goo.gl/XSO4QG
Very easy to understand and elegant way to present the different generations! ♥
Thanks for your kind comment, Philip! :)
Do lentivectors used in production of t-cell therapy cause false positive results in HIV testing?
Easy to understand. Thanks!
You're welcome!
Hello, Can you answer this question that if non-engineered lentivirus don't have psi gene for packing then what gene they use to do that ?
Very interesting video. Now 4 years after this video. Have there been breakthroughs? Just hoping
Absolutely! You can find more up-to-date information in our Knowledge Base: old.abmgood.com/marketing/knowledge_base/The_Lentivirus_System.php
I am trying to clone transcription factors such as sox2, klf4 and oct 4 and c-Myc using lentivirus and retrovirus in the fibroblast culture. I am very new in this field. can you guide me from where I should start it? I am naive in the cloning work....
Hi Ram, thanks for your comment!
First, determine what promoter would be best. For example, if you're planning on using your lentivirus/retrovirus for iPSC reprogramming, a good choice could be EF1a or PGF promoter. Once you know what promoter you'd like to use, you can go ahead and find a lentiviral/retroviral vector that includes the promoter you chose followed by a multiple cloning site (like our lentiviral cloning vector: goo.gl/bbGPFC).
Next, find and purchase the gene(s) you'd like to express (abm has a huge selection of genes in our ORF library: goo.gl/3PFduR).
Next, design primers to amplify each gene insert. You may want to design them with a ~15 bp overhang homologous to the vector arm and followed by a restriction enzyme site, then the regular binding portion. After amplification, you should have fragments that look like:
__vector arm homology__restriction site 1__GENE__restriction site 2__vector arm homology__
Next, you need to ligate your inserts to your lentiviral/retroviral vector. You'll first need to digest your vector using the two restriction enzymes. Follow this with seamless cloning (you can try our Pro Ligation-free cloning kit: goo.gl/2SHXVc) to ligate the fragments together. You'll need to do this for all the genes.
Finally, you'll need to package the resulting vector(s) into your lentivirus/retrovirus and then infect your fibroblast culture.
That's the gist of it in a nutshell!
There are easier routes, of course - you could just buy a premade iPSC lentivirus (we have lentiviruses available here: goo.gl/wCLSVm as well as retroviruses available here: goo.gl/e6Wgs1) that's ready to infect your cultures. Since you're working with sox2, klf4, oct4, and c-Myc, it may interest you to know we have all 4 of these genes available as pre-packaged lentiviruses already - either individually or as a set: goo.gl/HG9SEB
Cheers!
@@abmgood
This was a great explanation. I didn't quite understand the first-part about choosing the promoter. The link didn't work for me. I have a few questions.
What if I want my transgene to be expressed only by specific cells?
If there is a gene that I have identified let's say Gene X, where protein X is expressed by my desired group of cells (Alpha-Cells), how do I determine the right promoter to make sure I can express my transgene (gene Y) in Alpha-Cells only?
If you target embryonic cells or stem cells, how do you ensure only a subset of its differentiated progeny express the transgene?
Hi @@Matiasporrascool ! This is a challenging question to answer as it would depend on the cell type and whether you want expression ONLY in those cells and nothing else.
1) Controlling by delivery method:
For example, different serotypes (determined by the capsid of the virus) of AAV can infect different tissue types.
Eg. this table on our AAV page(www.abmgood.com/AAV-Adeno-Associated-Virus.html)
2) Using tissue-specific promoters, or genetic elements controlling expression levels or localization:
Specifically for alpha-cells, the best option would be to comb through the literature to see if there are any proven promoters that work specifically with alpha-cells.
Some resources that might be helpful are:
academic.oup.com/mend/article/30/2/248/2556546
link.springer.com/article/10.1007/s00424-016-1864-z
Hi,
4:28 "self-inactivation system is created as the deletion is transferred to the 5' LTR after reverse transcription and integration"
Could you give more detail regarding how the deletion can be transferred to 5' LTR?
Thanks for your comment, J Liu! In the life cycle of retroviruses, the U3 region of the 3' LTR is duplicated to form the corresponding region of the 5' LTR during the process of reverse transcription and viral DNA synthesis in infected cells. 133 bp in the U3 region of the 3' LTR, which contains the TATA box and binding sites for transcription factors Sp1 and NF-kB, is deleted and this gets transferred to the 5' LTR after reverse transcription. Consequently, the transcriptional unit from the LTRs in a provirus is eliminated. If you would like to know even more, here is reference ID showcasing the development of a self-inactivating lentivirus vector - PMID: 9733856. Hope this helps!
Very enlightenment video. Many thanks!
Glad to hear that you found the video helpful! :) Thanks for watching!
Hello. Would like to confirm if these recombinant lentivirus can infect an already infected CD4 Tcell especially those with reduced CD4 expression due to HIV accessory proteins.
I wonder that, why 293 T cells were used in Lentivirus Production?
Hi Ro, we use HEK 293T cells due to their ease of transfection, reliable growth rate, passaging ratios, and optimal size of cells. Furthermore, 293T cells have the "T" antigen (Hence 'T' addition from their parental HEK 293 cell line), which is crucial for lentivirus production to high copy number using plasmids that have an SV40 origin of replication (most lentiviral plasmids have this). Hope this helps!
Impressive so its viral replication is controlled by removing rna transcription factors and removing the dimer making further synthesis impossible. Then you're adding a broad range receptor surface for enhanced binding. But how is exact delivery determined then ? Wouldn't there be a possibility of nullifying or cross linking proteins that have ambiguity in binding receptor surfaces ? I just imagined the capsid surface would have a designer site specific receptor like one from an antigen or antibody.
Hello Fireteam Omega! Thanks for watching and leaving a comment! VSV-G has been shown to have broad cell tropism and lead to stable pseudotypes, making it a good choice for a wide range of applications. It's much simpler to use an already-existing coat protein with proven effectiveness in mediating viral entry than to custom-design a new coat protein. What determines the broader delivery is not that VSV-G binds to a greater variety of receptors than the env protein, but that VSV-G binds to one particular receptor family (the LDLR family), which is expressed widely in many cell types.
why we make a deletion u3 ltr
How does lenti compare to AAV for crispr knockout in human?
Either virus can be used to deliver sgRNA and Cas9, however, AAV's small packaging capacity means that it can only fit saCas9, not the more commonly used spCas9. AAV is preferred by some scientists due to its low immunogenicity and transient expression, especially for in vivo experiments. Lentivirus integrates to cause long-term expression, which can be useful for maximizing the possibility of success of the CRISPR experiment. However, some scientists worry that long-term expression of Cas9 might have unknown effects on their cell line. Hope this helps :)
Hello,
what's with inserting more than 1 transgene? What do I have to consider about? And how many transgenes will have the "offsprings"?
Hi Lina, the main concern when expressing multiple genes with lentivirus is packaging capacity. If you wish to express both genes from one vector, the total length of the sequence between the LTRs shouldn't exceed 9 kb. If your cassette would be larger than this, you may wish to package each gene separately. Another decision is whether you want to express the genes from the same promoter, but separated by an IRES or 2A peptide, or from two separate promoters.
I need to develop pseudovirus for RSV; Any technical assistantance?
Hello Ousman,
Please contact us at technical@abmgood.com if you need any technical assistance! Thank you.
Hi I tried with abm lenti vector with third generation packaging mix but GOI was not packaging into viral particles
Hi Suneetha, thank you for reaching out to us and we'd be happy to help you! Would you like to send your question to our experts in our technical team? You can email them directly: technical(AT)abmgood(DOT)com
Will Lentivirus spread to infect more cells? How effective is it to get to target tissue to have an effect with a single injection? Is does it need multiple injections?
Hi Justin, thanks for leaving your comment! All of our lentiviruses are replication incompetent - meaning they cannot replicate on their own to infect more cells once the virus is packaged. Single injection is highly effective, as Lentivirus has a relatively high transduction efficiency, and is stably integrating. Usually an MOI of 5-20 will work for any non-hard to transduce cell lines. Hope this helps!
@@abmgood yes thanks for the reply! Can lentivirus be used for multiple therapies? I know a built up immunity to AAV can be a problem. I'm wondering if there is the same problem with lentivirus.
@@JC-ly8pz Yes, it seems lentivirus can be used for multiple therapies, as immune responses in murine studies (either already existing, or acquired from multiple administrations) do not reduce the efficacy of lentiviral vectors! If you would like to read more, I would recommend this Paper in BMJ: thorax.bmj.com/content/72/2/137
is this process applicable for lenivirus simian virus also?
Hi Shayomi - thanks for leaving a comment! The process described is for applications of recombinant HIV Lentivirus systems in cell biology. That being said, there are 3rd generation systems for using SIV for gene delivery as well (though they are less commonly used) that are based on similar principles of safety and robust gene delivery. See this article on the use of SIV in Cystic Fibrosis gene therapie: goo.gl/VfD3ru
Thank you soo much for the rply keep up the good work👍
You're very welcome! Glad you enjoyed the videos!
why do I keep hearing losing my religion in this video, haha anyways I have to thank you it's very elaborated