Do you want to 'knock-down' a gene or 'knock-out' a gene? If you want to knock-out a gene to generate a null allele, you can opt for current tool such as CRISPR vectors, which can be obtained from Addgene.

Thank you very much for your kind suggestion, I want to Knock down my 3 desired gene of interest of my virus isolates through generating hpRNA. I got pKANNIBAL vector now but still i am requiring RNAi vector pART27 for final cloning of intermediate vector pKANNIBAL MCS cassettes. In addition, Can you please suggest which strain of E.coli will be more suitable to grow pKANNIBAL.

Hi Sanjeev,

1. I searched through some documents. Except for pHELLSGATE, due to its negative selection gene', which requires to be maintained in a specific E. Coli strain, DB3.1, there is no restriction on which E. Coli strain is needed for maintaining pKANNIBAL or pHANNIBAL (see attached paper). Probably you can try a common strain such as DH5a to see whether it works.

2. Why did you need pART27 for? It is a binary vector, people use it for Agrobacterium transformation for introducing RNAi into plants (see attached paper).

3. I attached the original paper creating pHANNIBAL, PKANNIBAL and PHELLSGATE for your reference.

Please also see the below links:

1. https://books.google.com/books?id=TYknr_Y20wwC&pg=PA127&lpg=PA127&dq=pKANNIBAL+and+competent+cells&source=bl&ots=IW6G5WFUEE&sig=J6Bl0LsimbfG_K4A31_oti-f9j0&hl=en&sa=X&ei=liFyVeWNGfWPsQTCyYIo&ved=0CD4Q6AEwBQ#v=onepage&q=pKANNIBAL%20and%20competent%20cells&f=false

2. https://books.google.com/books?id=jc2DT76CHRwC&pg=PA34&lpg=PA34&dq=pKANNIBAL+and+competent+cells&source=bl&ots=2J7Uv220NK&sig=jOAEdfXHUaIsfmFl6aMuPHvns8k&hl=en&sa=X&ei=liFyVeWNGfWPsQTCyYIo&ved=0CDoQ6AEwBA#v=onepage&q=pKANNIBAL%20and%20competent%20cells&f=false

Respected Yuan-Yeu Yau Sir,

Thank you very much for your kind suggestion, I tried to multiply the pKANNIBAL in DH5a, and that grows well. I am requiring the binary vector pART27 for final cloning of pcr products cloned in in pHANNIBAL/pKANNIBAL MCS cassettes through NotI restriction site. I searched some other vectors too which contains NotI in their MCS but didn't get anyone suitable and found in most of the literature that they have used pART27 vector.