G608G lamin A minigene
|Item||Catalog #||Description||Quantity||Price (USD)|
|Plasmid||20292||Standard format: Plasmid sent in bacteria as agar stab||1||$75|
This material is available to academics and nonprofits only.
- Backbone size w/o insert (bp) 4700
Vector typeMammalian Expression
Selectable markersNeomycin (select with G418)
Growth in Bacteria
Gene/Insert namelamin A
SpeciesH. sapiens (human)
MutationG606G:GGC>GGT Mutation causes aberrant splicing event, creating a mutant protein, delta50 lamin A, containing a 50 aa internal deletion in its globular tail domain.
Entrez GeneLMNA (a.k.a. CDCD1, CDDC, CMD1A, CMT2B1, EMD2, FPL, FPLD, FPLD2, HGPS, IDC, LDP1, LFP, LGMD1B, LMN1, LMNC, LMNL1, MADA, PRO1)
/ Fusion Protein
- GFP (N terminal on backbone)
- Cloning method Restriction Enzyme
- 5′ cloning site SacII (not destroyed)
- 3′ cloning site XbaI (not destroyed)
- 5′ sequencing primer EGFP-C (Common Sequencing Primers)
Terms and Licenses
- Not Available to Industry
To generate the wt lamin A minigene, intron 11 was first introduced between exon 11 and exon 12 in the lamin A cDNA in pEGFP-lamin A using a multi-step PCR reaction. In the fist step a region of the lamin A gene containing the last 156 bp of exon 11, intron 11 and the first 31 bp of exon 12 was amplified by PCR from genomic DNA obtained from HeLa cells, using the primers: LMNA-mini11wtF (5’-GCCCAGGTGGGCGGACCCATC-3’)/LMNA-mini12R (5’-CAGATTACATGATGCTGCAGTTCTG-3’). In the second step, a region of pEGFP-lamin A from the last 37 bp of exon 9 to the mutated region in exon 11 was amplified using the primers LMNA-mini9F/LMNA-mini11wtR (5’-GATGGGTCCGCCCACCTGGGC-3’). In the third step a region of pEGFP-lamin A containing the last 240 bp of exon 12 was amplified using the primers: LMNA-mini12F (5’-CAGAACTGCAGCATCATGTAATCTG-3’)/ GFPvectorR. The PCR products obtained from the first and third steps were mixed and used in a fourth PCR reaction in the presence of the primers LMNA-mini11wtF/GFPvectorR. The obtained PCR product was then mixed with the product obtained from the second step and used in a fifth PCR reaction with the primers: LMNA-mini9F/ GFPvectorR. The final amplification product was cloned into the BsiWI/XbaI site of pEGFP-lamin A to obtain wt pEGFP-lamin Aintr11. To generate the minigene wt pEGFP-lamin Aintr11 was digested with EcoRI and BstWI enzymes to remove a region of lamin A cDNA from exon 1 to the first 91 bp of exon 9, filled with Klenow enzyme and self-ligated. The mutated minigene was generated with the same procedure, substituting primers LMNA-mini11wtF e LMNA-mini11wtR respectively with: LMNA-mini11mutF (5’-GCCCAGGTGGGTGGACCCATC-3’) and LMNA-mini11mut R (5’-GATGGGTCCACCCACCTGGGC-3’).
These plasmids were created by your colleagues. Please acknowledge the Principal Investigator, cite the article in which the plasmids were described, and include Addgene in the Materials and Methods of your future publications.
For your Materials & Methods section:G608G lamin A minigene was a gift from Tom Misteli (Addgene plasmid # 20292 ; http://n2t.net/addgene:20292 ; RRID:Addgene_20292)
For your References section:Reversal of the cellular phenotype in the premature aging disease Hutchinson-Gilford progeria syndrome. Scaffidi P, Misteli T. Nat Med. 2005 Apr . 11(4):440-5. 10.1038/nm1204 PubMed 15750600