Creatine Transporter Deficiency examined using QRT-PCR of SLC6A8A and SLC6A8B knockout zebrafish embryos

Katherine Bassler, Joseph Sheheen, Dr. Susan C. Chapman

Abstract

Creatine Transporter Deficiency is an X-linked genetic disorder resulting in developmental delays, seizures, and intellectual disability. The disorder is caused by a mutation of the creatine transporter gene (SLC6A8). Creatine supplementation is partially effective in treating the muscular disorders related to the condition. However, the loss of the transporter in the blood brain barrier and in neuronal cells does not respond to supplementation. We are interested in the effect of SLC6A8 mutation on brain development. Zebrafish knockout models for both Slc6a8a and Slc6a8b paralogs, as well as wild-type controls, were bred and RNA isolated from the head for comparative analysis. Quantitative Real-Time PCR (qRT-PCR) was used to determine differential expression of neural patterning genes in both knockout lines compared to the wild-type controls. Here, we present the results from several sets of primers that have been developed to test the effect of Slc6a8a and Slc6a8b knockout: dlx2a, eomesa, lhx6, lim3, slc6a8a, slc6a8b, zash1a, with controls actb and gapdh.

Introduction

Creatine is an organic acid that acts as a phosphate donor required for effective recycling of ATP for the dynamic energy requirements in the brain. Creatine Transporter Deficiency is a result of a mutation in the creatine transporter, preventing creatine from entering cells [1]. Without the creatine transporter, creatine cannot pass through the blood brain barrier or enter neuronal cells, resulting in aberrant neural development. This disorder is an X-linked mutation in the SLC6A8 gene [2], and results in intellectual disability, delayed development of motor skills, and seizures.

qRT-PCR is used to measure the accumulation of amplification product in real time using a fluorescent reporter. In this study, we use qRT-PCR to determine the expression of several genes in zebrafish embryos, including both slc6a8a and slc6a8b knockout lines and in wild type controls. 

Materials and Methods

Primers for dlx2a, eomesa, lhx6, lim3, slc6a8a, slc6a8b, zash1a, and controls actb and gapdh were designed and purchased comercially (IDTDNA). Tissues were harvested from Slc6a8a, Slc6a8b, and AB control zebrafish embryos and stored in Trizol at -80℃. Nucleic acid quality was measured and quantified using Qubit and NanoDrop, as well as gel electrophoresis. RNA was extracted from zebrafish heads and whole embryos (Zymo) and diluted to a standardized concentration of 100 ng/µL, and split into 10 µL aliquots and stored at -80℃. For cDNA, 500 ng wildtype whole embryo RNA was converted using the iScript Reverse Transcription Supermix for Real Time PCR (Bio-rad). cDNA and the respective primers for each gene were amplified using PCR to prepare a standard. The standard for each gene was diluted to 1 ng/µL and a 5-point standard dilution was prepared at concentrations of 10-3 ng/µL through 10-7 ng/µL . Standards were prepared for each gene (eomesa, dlx2a, lhx6, lim3, slc6a8a, slc6a8b, zash1a, with gapdh and actb as controls) using wild type whole embryo cDNA and a forward and reverse primer, then were purified using a column. Concentration and quality were then determined using the NanoDrop and an agarose gel. The standard for each gene was diluted to 1 ng/ µL and tenfold dilutions were made to 10-7.  These were used to perform a 5-point standard curve and melt curve analysis for each gene using triplicate technical replicates and included one negative and two positive controls (Bio-rad CFX96 Touch RT-PCR detection system).

Results


Color key for amplification, melt curve, and melt peak graphs: 

10-3 ng/µL 

10-4 ng/µL 

10-5 ng/µL 

10-6 ng/µL 

10-7 ng/µL 

Positive Control (2 µL cDNA)

Positive Control (1 µL cDNA)

Negative Control (Water)


gapdh

Figure 1. Gapdh amplification, melt curve, and melt peak

Table 1. Melting temperatures of gapdh samples at 10-3 ng/µL through 10-7 ng/µL

Concentration (ng/µL)Replicate 1 Replicate 2 Replicate 3
10-381.00℃81.00℃81.00℃
10-481.00℃81.00℃81.00℃
10-581.00℃None81.00℃
10-681.00℃81.00℃81.00℃
10-780.50℃80.50℃80.50℃

actb

Figure 2. actb amplification, melt curve, and melt peak

Table 2. Melting temperatures of actb samples at 10-3 ng/µL through 10-7 ng/µL

Concentration (ng/µL)Replicate 1 Replicate 2 Replicate 3
10-382.00℃82.00℃82.00℃
10-482.00℃82.00℃81.50℃
10-582.00℃81.50℃81.50℃
10-681.50℃81.50℃81.50℃
10-781.50℃81.50℃81.50℃

dlx2a

Figure 3. dlx2a amplification, melt curve, and melt peak

Table 3. Melting temperatures of dlx2a samples at 10-3 ng/µL through 10-7 ng/µL

Concentration (ng/µL)Replicate 1 Replicate 2 Replicate 3
10-384.50℃84.50℃84.50℃
10-484.50℃84.50℃84.50℃
10-584.50℃84.50℃84.50℃
10-684.50℃84.50℃84.50℃
10-784.50℃84.50℃84.50℃

eomesa

Figure 4. eomesa amplification, melt curve, and melt peak

Table 4. Melting temperatures of eomesa samples at 10-3 ng/µL through 10-7 ng/µL

Concentration (ng/µL)Replicate 1 Replicate 2 Replicate 3
10-381.50℃81.50℃81.50℃
10-481.00℃81.00℃81.50℃
10-581.00℃81.50℃81.50℃
10-681.00℃81.00℃ + 85.00 ℃81.50℃
10-781.00℃81.00℃81.50℃

lhx6

Figure 5. lhx6 amplification, melt curve, and melt peak

Table 5. Melting temperatures of lhx6 samples at 10-3 ng/µL through 10-7 ng/µL

Concentration (ng/µL)Replicate 1 Replicate 2 Replicate 3
10-388.00℃88.00℃88.00℃
10-487.50℃87.50℃87.50℃
10-587.50℃87.50℃87.50℃
10-687.50℃87.50℃87.50℃
10-7NoneNone88.00℃

lim3

Figure 6. lim3 amplification, melt curve, and melt peak

Table 6. Melting temperatures of lim3 samples at 10-3 ng/µL through 10-7 ng/µL

Concentration (ng/µL)Replicate 1 Replicate 2 Replicate 3
10-387.00℃87.00℃86.50℃
10-486.50℃86.50℃86.50℃
10-586.50℃86.50℃86.50℃
10-686.50℃86.50℃86.50℃
10-786.50℃86.50℃86.50℃

slc6a8a

Concentration (ng/µL)Replicate 1 Replicate 2 Replicate 3
10-383.00℃83.00℃82.50℃
10-482.50℃82.50℃82.50℃
10-582.50℃82.50℃82.50℃
10-682.50℃82.50℃82.50℃
10-782.50℃82.50℃82.50℃

Figure 7. slc6a8a amplification, melt curve, and melt peak

Table 7. Melting temperatures of slc6a8a samples at 10-3 ng/µL through 10-7 ng/µL

slc6a8b

Figure 8. slc6a8b amplification, melt curve, and melt peak

Table 8. Melting temperatures of slc6a8b samples at 10-3 ng/µL through 10-7 ng/µL

Concentration (ng/µL)Replicate 1 Replicate 2 Replicate 3
10-384.50℃84.50℃84.50℃
10-484.00℃84.50℃84.50℃
10-584.00℃84.50℃84.50℃
10-684.00℃84.50℃84.50℃
10-784.00℃NoneNone

zash1a

Figure 9. slc6a8b amplification, melt curve, and melt peak

Table 9. Melting temperatures of slc6a8b samples at 10-3 ng/µL through 10-7 ng/µL

Concentration (ng/µL)Replicate 1 Replicate 2 Replicate 3
10-385.50℃85.50℃85.50℃
10-485.50℃85.50℃85.50℃
10-585.50℃85.50℃85.50℃
10-685.50℃85.50℃85.50℃
10-7NoneNone85.00℃

Conclusions

We have successfully performed qRT-PCR for seven genes (dlx2a, eomesa, lhx6, lim3, slc6a8a, slc6a8b, and zash1a) that have roles in patterning the developing zebrafish brain. Two control genes, actb and gapdh, were also successfully tested.
We have shown that we can harvest zebrafish tissues, make RNA and cDNA for use in qRT-PCR analysis.

Future Directions

We have completed harvesting head tissues from SLC6A8B knockout embryos and AB wildtype controls. SLC6A8A knockout embryos have been identified by genotyping and are being bred to provide tissues for RNA extraction. Once all tissues have been harvested and cDNA prepared, we will perform qRT-PCR analysis between the knockout embryos and control embryos to determine the effect of SLC6A8 mutations on zebrafish brain development.

Significance

Using zebrafish, we aim to determine which candidate patterning genes are affected during brain patterning when the creatine transporter is mutated. This will lead to a greater understanding of the effects of SLC6A8 mutations on human health outcomes.

References

  1. Mercimek-Mahmutoglu, S. Creatine Deficiency Syndromes https://www.ncbi.nlm.nih.gov/books/NBK3794/ (accessed Aug 18, 2019).
  2. SLC6A8 gene – Genetics Home Reference https://ghr.nlm.nih.gov/gene/SLC6A8 (accessed Aug 18, 2019).