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Results: 5

1.
Figure 4

Figure 4. Gga2 null mice exhibit low birthweights and hypoglycemia.. From: Analysis of Gga Null Mice Demonstrates a Non-Redundant Role for Mammalian GGA2 during Development.

A) Birthweights were obtained on day 1 and plotted according to genotype (wt, n = 42; Gga2+/−, n = 54, Gga2−/−, n = 12). * represents p<0.001; as determined by Student's t-test. B) Blood glucose levels in TIGM Gga2 newborn mice were determined on day 1 and plotted according to genotype (wt, n = 42; Gga2+/−, n = 54, Gga2−/−, n = 12) * represents p<0.001; as determined by Student's t-test. C) Blood glucose levels in Gga1 and Gga3 newborn mice were determined on day 1 and plotted according to genotype (wt, n = 10; Gga1−/−, n = 23; Gga3−/−, n = 20; Gga1−/−/3−/−, n = 20). * represents p<0.001, ** represents p<0.05; as determined by Student's t-test.

Jennifer Govero, et al. PLoS One. 2012;7(1):e30184.
2.
Figure 3

Figure 3. Analysis of Gga2 null mice (TIGM strain).. From: Analysis of Gga Null Mice Demonstrates a Non-Redundant Role for Mammalian GGA2 during Development.

A) The position of the gene-trap within intron-1 of the mouse Gga2 gene (cell line, IST10483E10) is shown. As a consequence of this gene-trap, normal messenger RNA (mRNA) is disrupted by the splice acceptor site of the engrailed-2 leader sequence fusing with codon-33 (encoding L33) of the mouse Gga2 gene. Gga2 primer sets fwd1/rev1 and fwd1a/rev2 (Figure S1A) were used to clearly distinguish between wt and null genotypes, respectively. B) PCR of genomic DNA was performed using Gga2 specific primers (reactions i) or a Gga2 specific primer in combination with a gene trap specific primer (reactions ii). PCR band in reactions (i) indicates wt genotype, while PCR band in reactions (ii) indicates null genotype. C) PCR was performed using genomic DNA from newborn pups of TIGM Gga2+/− crosses. 169 pups were analyzed and homozygous insertion of the gene-trap into the Gga2 gene was determined to be 95% lethal by 3 weeks of age. D) Western blot analysis of TIGM Gga2 mouse brain lysates probed for GGA1, GGA2, GGA3, AP-1 and GAPDH. * denotes a GGA1- cross reacting band (see Figure S4).

Jennifer Govero, et al. PLoS One. 2012;7(1):e30184.
3.
Figure 2

Figure 2. Analysis of Gga2 null mice (SYA176 strain).. From: Analysis of Gga Null Mice Demonstrates a Non-Redundant Role for Mammalian GGA2 during Development.

A) Insertion of the gene-trap cassette within intron-1 of the mouse Gga2 (SYA176) gene results in normal GGA2 mRNA being disrupted by the splice acceptor site of the engrailed-2 leader sequence fusing with codon-33 (encoding L33) of the mouse Gga2 gene. The progeny of Gga2+/− inter-crosses were genotyped by subjecting cDNA to RT-PCR with Gga2 primer sets fwd1/rev1 (spanning exons-1 through 5) and fwd1/rev2 (spanning exon-1 and the β-galactosidase exon) (Figure S1A), which differentiated between the wt and null genotypes, respectively. B) RT-PCR of cDNA prepared from white blood cells or brain tissue was performed using Gga2 specific primers (reactions i) or a Gga2 specific primer in combination with a gene trap specific primer (reactions ii). PCR band in reactions (i) indicates wt genotype, while PCR band in reactions (ii) indicates null genotype. C) Determination of the insertion site of the gene-trap within intron-1 of the mouse Gga2 gene (SYA176) allowed for the design of Gga2 primer sets fwd1/rev1 and fwd1/rev2 (Figure S1A), which clearly distinguished between wt and null genotypes, respectively, at the gene level. D) PCR of genomic DNA was performed using Gga2 specific primers (reactions i) or a Gga2 specific primer in combination with a gene trap specific primer (reactions ii). PCR band in reactions (i) indicates wt genotype, while PCR band in reactions (ii) indicates null genotype. E) The genotypes of the Gga2+/− crosses were determined either by RT-PCR (day1 pups) or Western blots (embryos E9, E10, E11 and E12). No homozygous null were obtained out of 162 newborns or embryos that were analyzed. F) PCR was performed using genomic DNA from newborn pups of SYA176 Gga2+/− crosses in the C57BL/6J background. No homozygous nulls were obtained out of 45 pups analyzed.

Jennifer Govero, et al. PLoS One. 2012;7(1):e30184.
4.
Figure 5

Figure 5. Tissue expression of GGA proteins.. From: Analysis of Gga Null Mice Demonstrates a Non-Redundant Role for Mammalian GGA2 during Development.

A) 25 µg of E9 whole embryo and E14, E18 and adult brain lysates along with 2–8 ng of purified standards were subjected to SDS-PAGE and immunoblot analysis. GGA1 and GGA2 purified standards were full-length Flag-tagged mouse proteins while the GGA3 standard was the mouse protein encoding only the VHS-GAT domain (see Figure S4A). Blots were probed for GGA1, GGA2, GGA3, AP-1 and GAPDH. Quantitation was derived from comparison to the GGA standards. # denotes a longer exposure of the GGA2 Western blot. B) The mRNA levels in mouse tissues were determined by quantitative realtime PCR and expressed relative to the expression of β-actin. First strand cDNA was synthesized from total RNA by reverse transcription as described in Materials & Methods. Realtime PCR was performed with SYBR Green Kit (ABI). The values presented are the average of two independent runs with total RNA isolated from the indicated organs of a wt mouse of C57BL/6J-129/Ola mixed genetic background. C) 25 µg of wt mouse brain lysates from mice (day 1 through adult stage) were subjected to SDS-PAGE and immunoblot analysis. Blots were probed as in panel A. D) 25 µg of wt adult mouse lysates (brain, liver, heart, lung and fat) were subjected to SDS-PAGE and immunoblot analysis. Blots were probed as in panel A.

Jennifer Govero, et al. PLoS One. 2012;7(1):e30184.
5.
Figure 1

Figure 1. Analysis of Gga1, Gga3 and Gga1/3 null mice.. From: Analysis of Gga Null Mice Demonstrates a Non-Redundant Role for Mammalian GGA2 during Development.

A) The position of the gene-trap within intron-7 of the mouse Gga1 gene is shown along with the position of the gene-trap within intron-1 of the mouse Gga3 gene. As a consequence of these gene-traps, normal mRNA is disrupted by the splice acceptor site of the engrailed-2 leader sequence fusing with either codon-203 (encoding E203) of the mouse Gga1 gene or codon-14 (encoding S14) of the mouse Gga3 gene. Gga1 and Gga3 PCR primer sets fwd1/rev1 and fwd1/rev2 (Figure S1A) were used to distinguish between wt and null genotypes, respectively. B) PCR of genomic DNA was performed using Gga1 specific primers, (Gga1 PCR, reaction i) Gga3 specific primers (Gga3 PCR, reaction i), and a Gga1 specific primer or Gga3 specific primer in combination with a gene trap specific primer (reactions ii). PCR band in reactions (i) indicates wt genotype, while PCR band in reactions (ii) indicates null genotype. C) Western blot analysis of mouse brain lysates probed for GGA1, GGA2, GGA3, AP-1 and GAPDH (loading control). * denotes a GGA1- cross reacting band (see Figure S4). D) Gga1+/−/Gga3−/−×Gga1+/−/Gga3−/− matings were set up and the resulting mice were genotyped by PCR as described in panels A and B. E) Birthweights were obtained on day 1 and plotted according to genotype (wt, n = 10; Gga1−/−, n = 23; Gga3−/−, n = 20; Gga1−/−/3−/−, n = 20). * represents p = <0.001; as determined by Student's t-test. F and G) Weights and nose-rump lengths (NRL) of wt (blue), Gga1−/− (red), Gga3−/− (yellow) and Gga1−/−/3−/− (green) mice were obtained at the times indicated and plotted versus age in weeks. * represents p<0.05, ** represents p<0.001; as determined by Student's t-test.

Jennifer Govero, et al. PLoS One. 2012;7(1):e30184.

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