Ron Lab


 

GCN2 knockout alleles

The targeting vector was constructed from PCR fragments amplified from cloned 129svev genomic DNA.

The 5' homology arm is a 6329 base pair genomic Xba1-EcoR1 fragment 147 base pairs 5' of exon 12. It was ligated into a pBS plasmid containing a thymidine kinase (TK) negative selection cassette. A double-stranded oligonucleotide pair with EcoR1 linkers containing a loxP site was introduced into the EcoR1 site. This 5' loxP site thus flanks exon 12 on its 5'. The 3' homology arm was recovered as a PCR fragment whose 5' end is the aforementioned genomic EcoR1 site and whose 3' end is in the 17th codon of exon 14 at a Kpn1 site introduced by the oligonucleotide used in the PCR. This 4023 base pair fragment was inserted at the EcoR1-Kpn1 sites of the aforementioned pBS plasmid. The loxP-flanked NeoR selection cassette was inserted into the intronic Nhe1 530 base pairs 3' of exon 12.

W4 ES cells were transfected with the targeting vector, linearized at the Kpn1 site and homologous recombination was confirmed by PCR and Southern blotting. The targeted ES cells were transfected transiently with a Cre-recombinase expression plasmids and derivative clones that had recombined across the 2 loxP sites flanking the NeoR selection cassette (GCN2.KO4c) as well as clones that had recombined across all three loxP sites (GCN2.K04ex) were isolated. The latter, GCN2.K04ex allele was used here. It deletes a 1101 base pair fragment that encompasses exon 12, which encodes the region of the kinase domain required for ATP binding. In addition, splicing of exon 11 to exon 13 is predicted to disrupt the reading frame of the mRNA and introduce multiple stop codons that destabilize the mRNA. Indeed no GCN2 signal is detected by in situ histohybridization or by immunoblot in brain tissues from GCN2.K04ex/ex mice using nucleic acid and antisera probes to regions of the mRNA and protein that are not encompassed by the deletion (see figure 1). The mutant allele is thus likely to be a null and is certain to direct no eIF2a kinase activity.

The mutant (known as GCN2.KO4ex) and corresponding wildtype alleles are detected by a three-primer PCR assay in which mGCN2.15S (TCT CCC AGC GGA ATC CGC ACA TCG) and mGCN2.4AS (AT CCA GGC GTT GTA GTA GCG CAC A) give a wildtype band of 374 bases and mGCN2.15S and mGCN218AS (T GCC ACT GTC AGA ATC TGA AGC AGG) give a 603 base-pair fragment from the deleted allele. The 1665 base pair fragment derived from the wildtype allele by amplification between mGCN2.15S and mGCN2.18AS is occasionally also detected in this assay.

Please note that the GCN2.KO cells are of the GCN2.KO4ex genotype which is detected by the three primer PCR described below.


Detecting GCN2.KO4ex genotype by PCR (07/01)

15S is a primer flanking the deletion on its 5' end.

4AS is a primer in the deleted segment

18AS is a primer flanking the deletion on its 3' end


Buffer

1.5mM MgCl2 (Fisher) or homemade 10x buffer (100mM Tris pH 8.3, 500mM KCl, 15mM MgCl2 )


Primers


mGCN2.15S (2x) 5' TCT CCC AGC GGA ATC CGC ACA TCG
mGCN2.18AS (1x) T GCC ACT GTC AGA ATC TGA AGC AGG
mGCN2.4AS (1x) 5' AT CCA GGC GTT GTA GTA GCG CAC A 3'


PCR Products

~374 bp fragment for WT with mGCN2.15S vs. mGCN2.4AS

~603 bp fragment for KO4EX (mut allele) with mGCN2.15S vs.mGCN2.18AS

(~1665 bp fragment for WT with mGCN2.15S vs. mGCN2.18AS)


PCR Conditions: (Long PCR)

94˚C, 4 min.

(94˚C, 10 sec.; 64˚C, 30 sec.; 68˚C, 4 min.) x 9 cycles

(94˚C, 10 min.; 64˚C, 30 sec.; 68˚C, 4 min. + 20 sec/cycle) x 20 cycles

72˚, 10 min

4˚, 30 min


RT

We have also created a conditional GCN2 allele in which loxP sites flank exon 12. The unexcised and excised versions of that allele GCN2.KO4c are detected with the PCR assay described below



Detecting GCN2.KO4c (Conditional) genotype by PCR (07/01)

Buffer

1.5mM MgCl2 (Fisher) or homemade 10x buffer (100mM Tris pH 8.3, 500mM KCl, 15mM MgCl2 )


Primers


mGCN2.15S 5' TCT CCC AGC GGA ATC CGC ACA TCG
mGCN2.4AS 5' AT CCA GGC GTT GTA GTA GCG CAC A 3'


PCR Products

~374 bp fragment for WT with mGCN2.15s vs. mGCN2.4AS

~413 bp fragment for KO4C with mGCN2.15S vs.mGCN2.4AS


PCR Conditions: (Long PCR)

94˚C, 4 min.

(94˚C, 10 sec.; 64˚C, 30 sec.; 68˚C, 4 min.) x 9 cycles

(94˚C, 10 min.; 64˚C, 30 sec.; 68˚C, 4 min. + 20 sec/cycle) x 20 cycles

72˚, 10 min

4˚, 30 min


RT

Download WORD document describing the construction of these alleles.