Cloning for expression analysis of ventricular genes co-expressed with myh7 in zebrafish

Congenital heart disease (CHD) is highly prevalent in live births and requires concise diagnoses and treatment plans [1]. Although there is a vast amount of cardiovascular genetics research in the field of CHD, there are still gaps in knowledge regarding the gene networks and cellular processes that drive heart development and function. Although many cardiac genes have been studied, there are still open questions surrounding the gene networks that regulate specific stages of heart development. Six genes with little to no previous expression data in the heart were selected for further molecular analysis. A previous single-cell RNA sequence showed that these genes were co-expressed with myh7 in the ventricle at 96 hours post fertilization (hpf). Forward and reverse primers were designed for each gene. To create a complementary DNA (cDNA) library, the total RNA was extracted from Danio Rerio (zebrafish) at 96 hpf. Using reverse transcription, mRNA was isolated and transcribed into cDNA. To amplify cDNA through polymerase chain reaction (PCR), forward and reverse gene-specific primers were used. Once we confirmed a successful PCR product via gel electrophoresis, we used bacterial transformation to clone the plasmid DNA. In order to isolate the plasmid DNA, minipreps will be used. We will then confirm isolation using a restriction digest and gel electrophoresis. Moving forward with this experiment, we will create RNA probes to conduct in-situ hybridization (ISH). When analyzing the temporal and spatial expression throughout heart development using ISH, we can draw conclusions about each gene’s potential functions during heart development. 

To identify new genes that function in the heart, single-cell RNA sequencing (scRNA-seq) was conducted on isolated zebrafish hearts at 96 hpf. The genes were categorized based on their co-expression with myh7, which is expressed in ventricular cardiomyocytes. Many genes that had no previously reported expression in the cardiovascular system were found to be co-expressed with myosin heavy chain 7 (myh7), suggesting that they are expressed in the ventricle. The expression patterns and functions of many of these genes have not been studied. From the genes found to be expressed in ventricular cardiomyocytes during scRNA-seq, we have selected dusp27, flnca, ndufa4a, lmod2b, myoz2b, and myom2a because their full expression patterns and functions have not been studied. Additionally, some of these genes are also linked to cardiovascular disease in humans. We hypothesize that these genes will be expressed in the cardiovascular system, specifically the ventricle, throughout early developmental stages. Since many of these genes are predicted to be involved in muscle contraction, we predict they will be involved in cardiomyocyte contractile function at 24 hours post fertilization (hpf), when the heart starts beating.

Table 1: Data on genes of interest

Dual specificity phosphatase 27 (abbreviation: dusp27) has reported expression in the nervous system and somites. It has reported gene function in skeletal myofibril assembly. dusp27 is significant because there's a mutant line with pericardial edema.

Filamin C, gamma a (abbreviation: flnca) has reported expression in the muscular system and somites. It has reported gene function in actin binding activity. flnca is significant because the gene is associated with hypertrophic cardiomyopathy in humans. 

NDUFA4 mitochondrial complex associated A (abbreviation: ndufa4a) has no previously reported expression data. It is expected to localize in the mitochondrial respiratory chain complex IV. ndufa4a mutations are predicted to lead to cardiomyopathy in humans. 

Leiomodin 2b (abbreviation: lmod2b) has no previously reported expression data. It is expected to be involved in muscle contraction. lmod2b is significant because it is orthologous to dilated cardiomyopathy in humans. 

Myozenin 2b (abbreviation: myoz2b) has reported expression in the muscular system. It is expected to be involved in actin binding activity. myoz2b is significant because it is orthologous to hypertrophic cardiomyopathy in humans. 

Myomesin 2a (abbreviation: myom2a) has no previously reported expression data. It is expected to have actin filament binding activity and be involved in cardiac muscle tissue morphogenesis. myom2a is significant because it is expected to be involved with cardiac muscle but has little prior studies. 

Designing primers: RNA sequences for each gene were taken from the National Center of Biotechnology Information. Primer 3 was used to aid designing the primers.  Forward and reverse primers were selected based on base pair size, lack of hairpins, and GC content, which determines the melting temperature.

Extraction for total RNA: Wild type zebrafish embryos were collected and placed in an incubator until 96 hpf. Once the fish reached 96 hpf, they were able to be fixed and homogenized. Purified total RNA was obtained from the homogenized tissues after a series of washes and DNase treatment. Gel electrophoresis and an RNA spec were used for quantification of total RNA.

Reverse transcription from messenger RNA: To isolate mRNA from total RNA extraction, oligo (dT) primer was used to bind to the poly-A at the 3’ end of the mRNA molecule. Reverse transcription from mRNA created complementary DNA (cDNA). 

Polymerase chain reaction: PCR undergoes denaturing, annealing, and extension to amplify a target DNA sequence, like our cDNA. PCR requires primers, dNTPs, Taq polymerase, and a DNA template. To confirm amplification of cDNA, gel electrophoresis was performed. When analyzing the gel electrophoresis, we looked for bands at a certain number of base pairs, based on the position of forward and reverse primers. 

Transformation and cloning: Obtained PCR product was transformed into a topoisomerase (TOPO) vector via heat shock. NEB 5-ɑ competent Escherichia Coli (E. coli) cells and TOPO vector were added to S.O.C. medium. Heat shock allows the plasmid DNA to enter the competent cells. Two volumes of transformation mixture were plated on ampicillin plates and incubated. 

Minipreps: To isolate plasmid DNA, minipreps were conducted. E. Coli bacterial colonies from ampicillin plates were inoculated in LB broth. Cultures were pelleted to separate bacterial cells from growth medium. Through a series of buffer washes, the plasmid DNA can be purified. To confirm DNA isolation, restriction digest and gel electrophoresis will be conducted.

See poster above:

Figure 1: Steps of research protocol.

Table 2: Results of total RNA spec quantification. Purified RNA should have 260 nanometers/280 nanometers ratio of around 2, as shown in both samples.

Figure 2: PCR amplification of cDNA gel electrophoresis results. Lanes 8 and 9 show correct flnca amplification. 

Lane 1: 1 kilobase ladder 

Lane 2: 100 base pair ladder  

Lanes 3 and 4: negative controls

Lanes 8 and 9: flnca band at 978 base pairs 

Figure 3: Bacterial transformation results. Two volumes of transformation mix were plated. Bacterial growth was observed on the 300 μL plate. Individual colonies were isolated for minipreps and further analysis.  

Figure 4: Expected final construct contains our gene of interest (flnca), forward promoter, coding sequence, and reverse promoter. T7 and SP6 RNA polymerase promoters initiate transcription. 

Moving forward, I plan on isolating the plasmid DNA by completing minipreps. Once the plasmid DNA is isolated, we can create RNA probes to complete in-situ hybridization (ISH). ISH shows temporal and spatial expression of particular genes. To further understand the expression patterns of our selected six genes, we will conduct ISH on whole embryos at multiple key stages throughout heart development. We hypothesize that our genes will be expressed early as 24 hpf, when the heart begins contracting.

Laboratory of Animal Resources

Waxman Lab at Cincinnati Children's Hospital

1. Pierpont ME, et al. Genetic basis for congenital heart defects: current knowledge: a scientific statement from the American Heart Association Congenital Cardiac Defects Committee, Council on Cardiovascular Disease in the Young: endorsed by the American