1. Deficiency of the Housekeeping Gene Hypoxanthine–Guanine Phosphoribosyltransferase (HPRT) Dysregulates Neurogenesis 
Ghiabe-Henri Guibinga, Stephen Hsu, Theodore Friedmann 
Molecular Therapy 
Volume 18, Issue 1, Pages (January 2010)
DOI: /mt
Copyright © 2010 The American Society of Gene & Cell Therapy Terms and Conditions

2. Figure 1
Expression of transcription factors required for specification and development of dopaminergic neurons, as determined by quantitative PCR. Cellular RNA purified from wild-type (open bars) and HPRT-deficient (closed bars) NT2 cells at 0 and 18 days of retinoic acid differentiation were examined by quantitative PCR for the transcription factors (a) Lmx1a, (b) Msx1, (c) Ngn2, (d) Mash1, (e) Nurr1, (f) Pitx3, and (g) FoxA1. * represents statistical significance between wild-type and HPRT-deficient cells (Student's t-test). bHLH, basic helix-loop-helix; HPRT, hypoxanthine–guanine phosphoribosyltransferase; mDA, midbrain dopaminergic neuron.
Molecular Therapy  , 54-62DOI: ( /mt )
Copyright © 2010 The American Society of Gene & Cell Therapy Terms and Conditions

3. Figure 2
Expression of transcription factors required for pan-neuronal development, as determined by quantitative PCR. Wild-type (open bars) and HPRT-deficient (closed bars). (a) Lmx1b, (b) Ngn1, (c) NeuroD, and (d) FoxA2. Gene expression on day 0 was at or near background levels for all four genes. * represents statistical significance between wild-type and HPRT-deficient cells (Student's t-test). bHLH, basic helix-loop-helix.
Molecular Therapy  , 54-62DOI: ( /mt )
Copyright © 2010 The American Society of Gene & Cell Therapy Terms and Conditions

4. Figure 3
Quantitative PCR analysis of gene expression of dopamine marker genes in undifferentiated (day 0) and differentiated (day 21) NT2 cells. Cellular RNA purified from wild-type (open bars) and HPRT-deficient (closed bars) NT2 cells at 0 and 21 days of retinoic acid differentiation was examined by quantitative PCR for each of the dopaminergic markers. * represents statistically significant difference between wild-type and HPRT-deficient cells (Student's t-test). AADC, aromatic L-amino-acid decarboxylase; TH, tyrosine hydroxylase.
Molecular Therapy  , 54-62DOI: ( /mt )
Copyright © 2010 The American Society of Gene & Cell Therapy Terms and Conditions

5. Figure 4
Analysis of neurite outgrowth in wild-type and HPRT-deficient NT2-derived neurons. (a) and (b) represent phase-contrast images of wild-type and HPRT-knockdown cells at 49 days, demonstrating blunted neurite outgrowth and rounded morphology of the latter cells. (c) Quantitation of neurite lengths in wild-type (open bars) and HPRT-knockdown cells (closed bars) at both 21 and 49 days. (d) Histogram of the frequency distribution of neurites in wild-type (open bars) and HPRT-deficient (closed bars) demonstrating a skewed distribution toward the shorter lengths in HPRT-deficient cells, especially evident at 49 days. The frequency distribution of neurite lengths was quantitated by MetaMorph imaging and morphometric and Matrox Intellicam software (see Materials and Methods). HPRT-Kd, hypoxanthine–guanine phosphoribosyltransferase-knockdown. * represents statistical significance between wild-type and HPRT-deficient cells (Student's t-test).
Molecular Therapy  , 54-62DOI: ( /mt )
Copyright © 2010 The American Society of Gene & Cell Therapy Terms and Conditions

6. Figure 5
Analysis of voltage-gated Na+ currents in wild-type and HPRT-deficient NT2-neuronal cells by whole cell patch clamp recordings. (a) Na+ currents (upper traces) were evoked by incremental 20 mV voltage steps (−60 to +40 mV, bottom traces) from a holding potential of −90 mV. The evoked Na+ currents could be totally abolished by the presence of 0.3 µmol/l TTX (middle traces). (b) The current to voltage (I–V) relation of the evoked Na+ currents was shown for wild-type (closed symbols) and HPRT-deficient (open symbols) NT2 cells on day 49 of RA-induced differentiation. The maximum current was obtained at −20 mV for both cell types. (c) Maximum Na+ currents were measured at 21 (open bars) and 49 days (filled bars) of RA induction in wild-type and HPRT-deficient NT2-neuronal cells. There were no reproducibly significant differences between the wild-type and HPRT-knockdown cells (n = 31). HPRT-Kd, hypoxanthine–guanine phosphoribosyltransferase-knockdown; TTX, tetrodotoxin.
Molecular Therapy  , 54-62DOI: ( /mt )
Copyright © 2010 The American Society of Gene & Cell Therapy Terms and Conditions