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Cloning and bacterial expression of postnatal mouse heart FGF-16
David P Sontag1, Peter A Cattini
1Division of Molecular Endocrinology, Department of Physiology, University of Manitoba, Winnipeg, Manitoba, Canada.
Abstract:
Fibroblast growth factor-16 (FGF-16) has been reported as the sixteenth member of the heparin sulphate proteoglycan binding growth factor family, which includes acidic and basic FGFs (FGF-1 and FGF-2), based on sequence similarity. The sequences of human (h) and rat (r) FGF-16 complimentary DNA (cDNA) sequences are known. Rat FGF-16 is expressed in brown adipose tissue during embryonic development but also shows some specificity for the postnatal heart. In spite of the importance of other FGF family members in cardiac physiology, there is scant information about FGF-16 function. As a first step towards exploiting mouse genetics in this regard, we have used reverse transcriptase-polymerase chain reaction and primers based on the rFGF-16 sequence to clone the adult mouse (m) FGF-16 cDNA. An mFGF-16 cDNA of 624 base pairs was generated. Based on sequence analysis, mFGF-16 and hFGF-16 share at least 95.2 and 99% nucleotide and amino acid similarity, respectively. In terms of other family members, FGF-16 is most closely related to FGF-9. When used as a radiolabeled probe, the mFGF-16 cDNA detected a single 1.8 kilobase transcript in adult mouse heart RNA. The mFGF-16 cDNA was also used to generate an amino-terminal poly-histidine tagged FGF-16 protein in bacteria. Using sodium dodecyl sulphate-polyacrylamide gel electrophoresis and taking into account the poly-histidine tag, an FGF-16 protein of 26.3 kDa was detected. The generation of cardiac mFGF-16 cDNA and a purified FGF-16 protein preparation are seen as important tools in the further characterization of FGF-16 expression and function in the mammalian heart.
Insights
Researchers cloned mouse Fibroblast Growth Factor-16 (FGF-16) cDNA and produced FGF-16 protein. This provides essential tools for studying FGF-16
Area of Science:
- Molecular Biology
- Cardiovascular Research
- Genetics
Background:
- Fibroblast Growth Factor-16 (FGF-16) is part of a growth factor family with known roles in cardiac physiology.
- Limited information exists regarding the specific function of FGF-16 in the heart.
- Understanding FGF-16 is crucial for advancing cardiac research.
Purpose of the Study:
- To clone the adult mouse FGF-16 cDNA.
- To generate a purified FGF-16 protein for further studies.
- To establish tools for investigating FGF-16 expression and function in the mammalian heart.
Main Methods:
- Reverse transcriptase-polymerase chain reaction (RT-PCR) using rat FGF-16 primers to amplify mouse FGF-16 cDNA.
- Sequence analysis to determine nucleotide and amino acid similarity.
- Northern blot analysis to detect FGF-16 transcripts in mouse heart RNA.
- Bacterial expression and purification of an amino-terminal poly-histidine tagged FGF-16 protein.
- Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) to confirm protein size.
Main Results:
- Successfully cloned a 624 base pair mouse FGF-16 cDNA.
- Mouse FGF-16 shares high nucleotide (95.2%) and amino acid (99%) similarity with human FGF-16.
- FGF-16 is most closely related to FGF-9 within the FGF family.
- Detected a single 1.8 kilobase transcript in adult mouse heart RNA.
- Generated a purified FGF-16 protein of 26.3 kDa.
Conclusions:
- The cloned mouse FGF-16 cDNA and purified protein are valuable resources.
- These tools will facilitate further characterization of FGF-16 expression and function in the mammalian heart.
- This research lays the groundwork for future genetic studies on FGF-16 in cardiac physiology.