cDNA

HOXA9-MSCV-CD24

20975

HOXA9-MSCV-CD24

Ampicillin

*Please note that the full sequence verified by Addgene differs from the original depositor's map and thus the numbers and nomenclature used by the depositor may differ from the Addgene map.* Depositor's Comments: We discovered that, although a 1,975-nt full-length Hoxa9 clone (numbered from the protein start site; I.M.A.G.E. clone BC055059) would produce protein when transiently expressed from the murine stem cell virus (MSCV) long terminal repeat, following retroviral transduction, the HOXA9 protein could not be detected. Since this cDNA contained two putative polyadenylation signals in the 3' UTR, we removed these sites to determine if they were interfering with retrovirally mediated protein expression. The site at nt 1798 was replaced using a primer containing a KpnI sequence in place of the AATAAA sequence together with a primer containing the BglII site at nt 749 to amplify a 1,052-nt BglII-KpnI fragment by PCR. A second primer containing the KpnI site in place of the polyadenylation signal at nt 1798 was used in conjunction with a primer containing an AAT-to-CGG change in the polyadenylation signal at nt 1975 and an adjacent XhoI site for cloning to generate a 150-nt KpnI-XhoI fragment. Standard methods were used to combine these fragments to create a 1,975-nt Hoxa9 cDNA encoding an N-terminal FLAG-tagged version of the mature protein but lacking the two putative polyadenylation signals. The resulting MSCV clone produced high levels of FLAG-HOXA9 protein and immortalized BM cells.

37

Debbie Wolgemuth

High Copy