HOXA9-MSCV-CD24
(Plasmid #20975)

• Depositing Lab: Corey Largman
• Publication: Shen et al Mol Cell Biol. 2008 Jul . 28(14):4609-19.

Backbone

• Vector backbone: MSCV (murine stem cell virus)
• Total vector size (bp): 7842
• Vector type: Mammalian Expression, Retroviral

Growth in Bacteria

• Bacterial Resistance(s): Ampicillin, 100 μg/mL
• Growth Temperature: 37°C
• Growth Strain(s): DH5alpha
• Copy number: High Copy

Gene/Insert 1

• Gene/Insert name: HOXA9
• Species: M. musculus (mouse)
• Mutation: Full length cDNA with ~1.1kb 3'UTR with 2 putative polyA signals mutated to allow expression in MSCV+CD24 selection epitope (FACS surface marker). No polyA signal.
• Entrez Gene: Hoxa9 (a.k.a. D6a9, Hox-1.7)
• Tag / Fusion Protein:
  • Flag (N terminal on backbone)

Cloning Information for Gene/Insert 1

• Cloning method: Restriction Enzyme
• 5′ cloning site: EcoRI (not destroyed)
• 3′ cloning site: XhoI (not destroyed)
• 5′ sequencing primer: MSCV

Gene/Insert 2

• Gene/Insert name: CD24
• Alt name: CD24 isoform a preproprotein NP_001278666.1
• Species: H. sapiens (human)
• GenBank ID: NP_001278666.1
• Entrez Gene: CD24 (a.k.a. CD24A)

Resource Information

• A portion of this plasmid was derived from a plasmid made by: Debbie Wolgemuth

Terms and Licenses

• Academic/Nonprofit Terms:
  • UBMTA
• Industry Terms:
  • Not Available to Industry

Depositor Comments

*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.