Biomedical subjects

D C McFarland

Publications and source records attributed to D C McFarland.

At least 19 recordsLinked to original sources

PubMed2006

Effect of transforming growth factor-beta on decorin and beta1 integrin expression during muscle development in chickens.

Myoblast-extracellular matrix interactions play a pivotal role in skeletal muscle development. Transforming growth factor-beta (TGF-beta) is a key regulator of muscle cell proliferation and differentiation. The level of TGF-beta expressed will affect the concentration of the extracellular matrix proteoglycan decorin and the cell surface beta1 integrin subunit. The decorin proteoglycan is a regulator of cell growth as well as the organization of the extracellular matrix. The beta1 integrin plays a role in muscle cell attachment, migration, and the formation of multinucleated myotubes. In the current study, chicken myogenic satellite cells isolated from the pectoralis major muscle from the chicken genetic muscle weakness, low score normal (LSN), and normal pectoralis major muscle were used to investigate TGF-beta expression as it relates to decorin and beta1 integrin mRNA expression. The LSN muscle defect is characterized by altered myotube formation and sarcomere structure, and the satellite cells have reduced proliferation and differentiation. The mRNA expression was measured by real-time quantitative reverse transcription PCR. The LSN condition has elevated expression of TGF-beta2 and TGF-beta4 with increased expression of decorin and decreased beta1 integrin during myogenic satellite cell proliferation and differentiation. Normal satellite cell cultures were treated with the addition of exogenous TGF-beta during differentiation to determine if the altered expression of LSN decorin and beta1 integrin was associated with TGF-beta expression. The addition of exogenous TGF-beta decreased decorin expression during differentiation and reduced beta1 integrin expression at 24 and 48 h of differentiation. These results suggested that alteration of decorin expression in the LSN myogenic satellite cells may occur by a mechanism involving factors in addition to TGF-beta, but the addition of exogenous TGF-beta did affect both decorin and beta1 integrin expression. These data, therefore, suggested that TGF-beta might play a pivotal role in chicken skeletal muscle formation through modulation of the expression of both extracellular matrix molecules and cellular receptors important in the control of cell migration and growth regulation.

Animals↗

PubMed2006

Differential expression of membrane-associated heparan sulfate proteoglycans in the skeletal muscle of turkeys with different growth rates.

Heparan sulfate proteoglycans (HSPG) are key components of the cell membrane and extracellular matrix of skeletal muscle cells. Two major groups of membrane-associated HSPG found in skeletal muscle are syndecans (SYN) and glypicans (GPC), both of which can regulate growth factor activities and, thus, modulate cell proliferation and differentiation. In the current study, the mRNA expression of a group of membrane-associated HSPG (SYN 2 through 4 and GPC 1) was investigated in embryonic pectoralis major muscle [embryonic days (ED) 14, 16, 18, 20, 22, 24] and myogenic satellite cells isolated from males of a turkey genetic line selected for increased 16-wk BW (F line) and an unselected randombred control (RBC2 line) from which the F line was developed. The mRNA expression was measured by a real-time quantitative PCR approach. The SYN 2 and SYN 4 expression exhibited a similar pattern during embryonic p. major muscle development, which remained constant from ED 14 to ED 22 and declined sharply from ED 22 to ED 24 to a very low level. In contrast, the SYN 3 and GPC 1 expression showed a continuous decline from ED 14 to ED 24. The F line had higher SYN 2 (ED 14, 18, 20, 22), SYN 3 (ED 22), and SYN 4 (ED 22) expression than the RBC2 line. In myogenic satellite cells, initiating differentiation resulted in a decrease in SYN 2 expression and an increase in GPC 1 expression. Both SYN 3 and SYN 4 expression stayed almost constant through both the proliferation and differentiation stages. The proliferating satellite cells from the F line displayed higher SYN 4 expression than those from the RBC2 line. Collectively, the results from the current study suggest that membrane-associated HSPG are differentially expressed in both embryonic p. major muscle tissue and satellite cells isolated from F-line and RBC2-line male turkeys, implying their distinct roles in myogenesis and differing influence on muscle growth properties.

Aging↗

PubMed2005

Effect of genetic selection on MyoD and myogenin expression in turkeys with different growth rates.

Skeletal muscle development requires the ordered expression of specific myogenic regulatory factors, which include MyoD, Myf5, myogenin, and MRF4. The MyoD and Mrf5 factors are required for the determination of myoblasts, whereas myogenin and MRF4 play a pivotal role in terminal differentiation. In the current study, males and females of a turkey genetic line selected only for increased 16-wk BW (F line) and an unselected randombred control (RBC2 line) from which the F line was developed were used to investigate the developmental expression of MyoD and myogenin mRNA in embryonic pectoralis major muscle and myogenic satellite cells. Pectoralis major muscle was isolated at embryonic d (ED) 14, 16, 18, 20, 22, and 24. The mRNA levels of MyoD and myogenin were measured using a real-time quantitative polymerase chain reaction method. Both MyoD and myogenin expression declined during embryonic development. The decrease in MyoD expression started at ED 16 for the F line and at ED 18 for the RBC2 line for both sexes. Myogenin expression in both lines began to decline at ED 14. The F line males had lower myogenin expression at ED 14, 16, and 18 than the RBC2 line males, which was similar for the F line females compared with the RBC2 line females except there was no significant difference at ED 18. The RBC2 line males had greater expression than the females for myogenin at ED 16 and 18 for the RBC2 line. Proliferating myogenic satellite cells in both lines and sexes expressed low levels of MyoD and myogenin. After the initiation of differentiation in both lines and sexes, there was a sharp surge in MyoD expression at 24 h followed by a decrease at 48 h and then an increase in expression through 72 or 96 h of differentiation. There were line and sex differences in myogenin expression during the differentiation process. These data are suggestive of growth- and sex-related differences in the expression of myogenic regulatory factors key to muscle cell proliferation and differentiation, which will affect muscle growth rate.

Animals↗

PubMed2004

Beta1 integrin mediation of myogenic differentiation: implications for satellite cell differentiation.

Myoblast-extracellular matrix interactions mediated by integrin receptors have been shown to play a pivotal role in skeletal muscle development. In the current study, the avian genetic muscle weakness, low score normal (LSN), which exhibits modified myotube and sarcomere structure and a reduction in beta1 integrin expression during satellite cell differentiation, was used as a model system to further investigate the role of beta1 integrins in myogenic differentiation. During normal satellite cell differentiation, the beta1 integrin was localized at areas of pseudopodial activity and points of cell contact. In contrast, in LSN satellite cell cultures the beta1 integrin was predominantly observed in clusters not associated with points of cell to cell contact. The LSN satellite cells underwent apoptosis at the same time normal satellite cells were fusing to form multinucleated myotubes. During muscle development, the beta1 integrin is regulated by alternative splicing from a beta1A to beta1D form. During both embryonic and posthatch pectoralis major muscle development, regulation of the beta1A and beta1D variants was altered in the LSN. Normal and LSN myogenic satellite cell cultures did not express the beta1D integrin, but the LSN satellite cells expressed higher concentrations of the beta1A integrin than did normal satellite cells. Taken together, these data further demonstrate the importance of the beta1 integrin subunit in the regulation of cell-extracellular matrix signal transduction pathways during muscle growth and differentiation.

Amino Acid Sequence↗

PubMed2004

Effects of syndecan-1 and glypican on muscle cell proliferation and differentiation: implications for possible functions during myogenesis.

The heparan sulfate proteoglycans, syndecan-1 and glypican, are low-affinity receptors for fibroblast growth factor 2 (FGF2). Because FGF2 is a potent stimulator of skeletal muscle cell proliferation and a strong inhibitor of differentiation, it is likely that changes in syndecan-1 and glypican expression will affect myogenesis as both, in part, regulate FGF-dependent signaling. In the current study, expression vector constructs containing either syndecan-1 or glypican were transfected into turkey myogenic satellite cells resulting in the overexpression of these genes. The amount of expression of each of these genes was measured by semiquantitative reverse transcriptase polymerase chain reaction. The satellite cell cultures overexpressing syndecan-1 were unable to fuse to form multinucleated myotubes after differentiation was induced. The syndecan-1-transfected cells maintained a rounded morphology typical of cells during proliferation. In contrast, the satellite cells transfected with glypican formed larger myotubes. These results suggest that both syndecan-1 and glypican play pivotal, but different, roles in both muscle cell proliferation and differentiation.

Animals↗

PubMed2003

Expression of fibroblast growth factor 2 and its receptor during skeletal muscle development from turkeys with different growth rates.

Fibroblast growth factor 2 (FGF2) is a key regulator of muscle cell proliferation and differentiation. To address how FGF2 and fibroblast growth factor receptor 1 (FGFR1) gene expression influences skeletal muscle development and growth, pectoralis major muscle was isolated at embryonic days (ED) 14, 16, 18, 20, 22, and 24, and at 1-, 8-, 12-, and 16-week posthatch from a turkey line (F) selected only for increased 16-week body weight and its genetic control line (RBC2). The mRNA levels of FGF2 and FGFR1 were measured by semi-quantitative reverse transcription polymerase chain reaction. Compared to the RBC2 line males, the F line males had higher FGF2 mRNA levels at ED 14 and 16, and higher FGFR1 mRNA levels at ED 18, but down-regulated FGF2 and FGFR1 gene expression at ED 22. Although no FGF2 mRNA was detected in posthatch muscle tissue, the F line turkeys had more FGFR1 gene expression at 8-, 12-, and 16-week posthatch than the RBC2 line turkeys. During myogenic satellite cell proliferation, the F line cells had higher FGF2 and FGFR1 mRNA levels than the RBC2 line cells. The satellite cell responsiveness to FGF2 treatment was evaluated by the ability of the cells to proliferate. The male satellite cells were more responsive to FGF2 stimulation than the female cells in both lines. These results suggest that the F line turkeys have increased FGF2 signaling that may affect muscle cell proliferation and differentiation, which may also lead to an enhancement in muscle development and growth rate.

Animals↗

PubMed2003

Ventricular myosin heavy chain isoform expression is altered in vitro in low score normal chickens.

The low score normal (LSN) chicken exhibits a genetic muscle weakness and altered in vitro myogenesis compared to the normal White Leghorn chicken. The ventricular myosin heavy chain isoform has been reported to be the initial muscle-specific contractile protein expressed during myogenesis. The goals of this study were to determine whether altered myogenesis of the LSN satellite cells in culture was accompanied by delayed ventricular myosin heavy chain expression and to further characterize the altered myogenic events exhibited by the LSN chicken. Immunocytochemical and ELISA analyses were employed to document the temporal expression of the ventricular myosin heavy chain during LSN chicken myogenesis. Satellite cells derived from the LSN chicken pectoralis major exhibited lower (P </= 0.05) expression of ventricular myosin heavy chain during proliferation and differentiation in culture than did satellite cells derived from White Leghorn chickens. Low score normal cells failing to express the ventricular myosin heavy chain generally remained mononucleated and unfused, whereas cells that were multinucleated appeared to express ventricular myosin heavy chain regardless of the avian source. These results suggest that the altered myogenesis observed in LSN chickens is associated with delayed ventricular myosin heavy chain expression.

Animals↗

PubMed2002

Developmental expression of skeletal muscle heparan sulfate proteoglycans in turkeys with different growth rates.

Heparan sulfate proteoglycans (HSPG) are a group of extracellular matrix molecules that link skeletal muscle cells to their extrinsic environment. To investigate if HSPG expression is affected by muscle growth and gender, a turkey line (F) selected for increased 16-wk BW and its unselected random-bred control line, RBC2, were used in the present study. Heparan sulfate (HS) and HSPG levels were measured in embryonic and posthatch pectoralis major muscle. HS levels plateaued at embryonic day (ED) 16 in both lines. A significant decrease of HS occurred at ED 18 in F males and females, and at ED 20 and 22 in the RBC2 males and females, respectively. Embryonic HSPG levels peaked at ED 18, and were significantly higher from ED 14 through 18 in F males and females compared with those of the RBC2 line. Male pectoralis major muscle had more HSPG at early embryonic stages than female muscle in both lines. During 1 to 16 wk posthatch, F male and female pectoralis major muscle contained more HSPG than the RBC2 samples, and HSPG levels in F males were higher than those of the females. Myogenic satellite cells derived from F and RBC2 male and female pectoralis major muscle were cultured to measure HSPG expression during proliferation and differentiation. No significant difference in HSPG level was found between the RBC2 and F line cells. However, in both lines, male-derived satellite cells had more HSPG than the female cells during proliferation and differentiation. These data show that HS and HSPG expression are affected by muscle growth properties and sex.

Age Factors↗

PubMed2002

The effect of hepatocyte growth factor on turkey satellite cell proliferation and differentiation.

The effect of hepatocyte growth factor (HGF) on turkey satellite cell proliferation and differentiation was examined in cell culture. Satellite cell clones were established from one muscle of an individual turkey. The results showed that HGF is a potent activator and mitogen of turkey satellite cells and embryonic myoblasts with maximal stimulation at 1 ng/mL. HGF is also an inhibitor of differentiation of turkey satellite cells. Heterogeneity in the responsiveness to HGF in the turkey satellite cell population was observed between clones selected for fast (Early) or slow (Late) rates of proliferation. However, two other Early clones exhibited responses similar to those of two other Late clones. When combined with insulin-like growth factor (IGF) and fibroblast growth factor (FGF), singularly or in combination, HGF did not exert any additive or synergistic effects on Early or Late clone proliferation. Whereas when combined with IGF, FGF, and platelet-derived growth factor (PDGF), HGF significantly stimulated proliferation of the Late clone but not the Early clone. Addition of anti-HGF antibody to culture media diminished proliferation and provided evidence of autocrine production of HGF by turkey satellite cell cultures. Heterogeneity also exists in the turkey satellite cell population with respect to autocrine production of HGF.

Animals↗

PubMed2001

Temporal expression of growth factor genes during myogenesis of satellite cells derived from the biceps femoris and pectoralis major muscles of the chicken.

The expression of mRNAs for transforming growth factors (TGF-beta2, myostatin, activin-B, and follistatin), insulin-like growth factors (IGF-I and -II), and fibroblast growth factor (basic, bFGF) was investigated in satellite cells derived from chicken pectoralis major (PM) and biceps femoris (BF) muscles in the stages from initiation of proliferation to fusion. These growth factor gene cDNAs were synthesized by reverse transcriptase polymerase chain reaction (RT-PCR). No myostatin, activin-B, follistatin or bFGF expression was detected in either cell culture at 0 h. TGF-beta2 mRNA level increased at 48 h (P < 0.01) and remained constant through 144 h in both PM and BF satellite cell cultures. The ontogeny of myostatin gene expression with the exception of a sharp increase in BF culture at 72 h (P < 0.01), was nearly identical in both cell cultures. Activin-B mRNA level in PM satellite cells was higher than that in BF satellite cells at 72 h and 120 h (P < 0.01). Follistatin mRNA in PM satellite cells was higher than that in BF satellite cells at 24, 96, and 120 h culture (P < 0.01). No IGF-I gene expression was detected in cell cultures at any time point. IGF-II gene expression in BF satellite cells declined at 96 h (P < 0.01) and remained reduced until 144 h. bFGF mRNA in both satellite cell cultures increased at 24 h (P < 0.05) and remained at this level in BF satellite cells through 144 h.

Actins↗

PubMed2001

The effect of insulin-like growth factor analogs on turkey satellite cell and embryonic myoblast proliferation.

The effects of several human and chicken insulin-like growth factor (IGF) analogs on turkey satellite cell and embryonic myoblast proliferation were examined in serum-free medium. Similar rates of proliferation were observed when human or chicken IGF-I or IGF-II (13.1 nM) was administered to satellite cells. The biopotency of two analogs, which were modified to prevent interaction with IGF-binding proteins, was also examined. Human Des(1-6)IGF-II was equipotent to native human and chicken IGF-II. However, the chicken LR3 IGF-I analog was significantly less active toward satellite cells and embryonic myoblasts compared with chicken IGF-I. Human [Leu27] IGF-II, an analog designed to have reduced affinity to the IGF Type I receptor but unaltered binding to IGF-binding proteins, had a diminished effect on cell proliferation. Examination of IGF receptor binding characteristics revealed that chicken LR3 IGF-I had reduced ability to compete with [125I]hIGF-I for binding to satellite cells or embryonic myoblasts compared with chicken IGF-I. The observed biological responses to IGF suggest that IGF-binding proteins have little effect on Type I IGF receptor action in these cell types in serum-free medium. The results also suggest that alterations of the IGF molecule to prevent interaction with binding proteins may also alter receptor binding affinity.

Animals↗

PubMed2000

Telomeric profiles and telomerase activity in turkey satellite cell clones with different in vitro growth characteristics.

The satellite cell population in postnatal skeletal muscle is heterogeneous because individual satellite cells isolated from a single muscle have differing abilities to proliferate under the same in vitro conditions. Telomeres are structures found at the ends of all eukaryotic chromosomes that are characterized by repetitive DNA sequences, and they are important in determining cellular proliferation potential. The relationship between satellite cell proliferative heterogeneity and telomeric DNA was examined by digesting genomic DNA from large-colony-forming and small-colony-forming turkey satellite cell clones with HinfI, separating the restriction fragments on an agarose gel, and hybridizing the gels with an oligonucleotide probe specific for telomeric DNA. Turkey satellite cells generated telomeric restriction fragments up to approximately 180 kB. The large-colony-forming satellite cell clones had a larger proportion (P<0.05) of total telomeric restriction fragments below 33 kB than the small-colony-forming satellite cell clones. However, telomerase expression was detected in cultures from large-colony-forming and small-colony-forming turkey satellite cells suggesting that the differences in telomeric restriction fragments may not be related to the differences in in vitro proliferative behavior and that telomerase may contribute to the high in vitro growth capacity of turkey satellite cells.

Animals↗

PubMed2000

Heterogeneity in growth and differentiation characteristics in male and female satellite cells isolated from turkey lines with different growth rates.

The effects of growth- and gender-related differences on satellite cell proliferation and differentiation were investigated using satellite cells isolated from the pectoralis major muscle of a turkey line selected for increased 16-week body weight (F-line) and its unselected randombred control (RBC2-line). Proliferation rates within the F- and RBC2-lines did not differ between sexes. The F-line male and female satellite cells when compared to the RBC2-line male and female satellite cells proliferated at a faster rate. Differentiation rates were increased for the F-line male cells compared to both the F-line female and RBC2-line male satellite cells. No difference in differentiation rate was noted within the RBC2-line satellite cells. For satellite cells from females, the RBC2-line differentiated faster than the F-line. Morphological data on myotube length and the number of nuclei per myotube supported the differentiation data in that F-line male satellite cells had the longest myotubes with the most nuclei, there was no significant difference between myotubes within the RBC2-line, and female-derived myotubes from the RBC2-line were longer than those of the F-line by 96 h of fusion. These data are suggestive of both growth- and gender- related differences in satellite cell proliferation and differentiation.

Animals↗

PubMed2000

Preparation of pure cell cultures by cloning.

Due to the heterogeneous nature of animal organs, primary cell cultures potentially contain more than one type of cell. Interpretation of data arising from studies using these mixed cultures can lead to difficulties, because it may not be possible to ascertain which of the cell types present may have responded to a given treatment. While a number of procedures have been used to enrich the cell population of interest, many scientists have resorted to cloning of cells in order to insure the purity of cell cultures. Three major strategies have been used to produce clones, namely, the dilution technique, cloning ring technique, and robotic cell transfer. Successful cloning is dependent on optimization of attachment substrata, basal media composition, serum source, and growth factor/hormone additions to support proliferation of the cell type at clonal (low) density.

Animals↗

PubMed2000

Isolation and characterization of myogenic satellite cells from the muscular dystrophic hamster.

Myogenic satellite cells were isolated from control and dystrophic hamster diaphragms to examine cellular mechanisms involved in the physiology of muscular dystrophy. The Bio 14.6 dystrophic hamster, which possesses a defect in the delta-sarcoglycan gene, develops biochemical and physical symptoms of Duchenne-like and limb girdle muscular dystrophies. Because primary cultures of the control and dystrophic satellite cells became extensively contaminated with non-myogenic cells during proliferation, cell clones were developed to provide pure cultures for study. Cell culture conditions were optimized with the use of Ham's F-12K medium containing 10% fetal bovine serum +5% horse serum + 10 ng/mL basic fibroblast growth factor + 50 microg/mL porcine gelatin. Proliferation rates of the two clonal cultures were similar between the two lines. Satellite cell-derived myotubes from both primary cultures and clones differed between control and dystrophic animals. Dystrophic myotubes tended to be long and narrow, while the control-derived myotubes were broader. Measurement of muscle-specific creatine kinase during differentiation revealed that the dystrophic myotubes possessed higher creatine kinase levels than control myotubes (up to 146-fold at 168 h). The results demonstrate that satellite cells can be isolated from the hamster and may provide a useful tool to study muscular dystrophies associated with defects in the sarcoglycan complex and the involvement of sarcoglycans in normal skeletal muscle growth and development.

Animals↗

PubMed2000

Beta1 integrin expression during normal and low score normal avian myogenesis.

Skeletal muscle development is, in part, regulated by myoblast-extracellular matrix interactions mediated by the transmembrane integrin family of heterodimeric receptors. The avian genetic muscle weakness, low score normal (LSN), exhibits modified myotube and sarcomere structure that may be associated with altered integrin expression. Protein expression of the beta1 integrin subunit was measured during normal and LSN Pectoralis major muscle development at 14, 16, and 18 d of embryonic development, and 1 d and 1 and 6 wk posthatch. During embryonic development, integrin expression was downregulated. However, by 1 wk posthatch, integrin levels were upregulated and remained elevated through 6 wk posthatch. This pattern was observed in both normal and LSN muscle development. Overall, beta1 integrin levels were lower in the LSN P. major muscle. In normal and LSN satellite cell cultures, beta1 integrin expression was low during proliferation. In early differentiation, beta1 integrin expression increased and was then downregulated. As observed in the muscle extracts, LSN beta1 integrin expression was significantly lower during differentiation. These results suggest that the regulation of beta1 integrin expression is critical to the progression of myogenesis, and, during LSN myogenesis, decreased expression of beta1 integrin may be associated with modifications in muscle structure.

Animals↗

PubMed2000

Response of C2C12 mouse and turkey skeletal muscle cells to the beta-adrenergic agonist ractopamine.

The effects of ractopamine (RAC) and ractopamine stereoisomers (RR, RS, SR, and SS) on cyclic AMP (cAMP) production, total protein, and DNA concentrations in mouse skeletal muscle cells (C2C12) were evaluated. The RAC (10 microM) caused an approximately 30% increase in cell number, protein, and DNA concentrations in myoblasts after 48 h; no differences were found in myotubes. The RAC-stimulated increase of these variables in myoblasts was blocked by the presence of equimolar concentrations of propranolol. At a later passage, myoblasts failed to exhibit an increase in cell number, protein, or DNA upon exposure to RAC. Both myoblasts and myotubes increased cAMP production in response to 10 microM RAC. The RAC isomers ranked RR >> SR > RS approximately SS in ability to stimulate cAMP production, with essentially no response to SS. The SR produced about 50% of the RR response. Coincubation of propranolol (10 microM) and RAC (10 microM) prevented RAC-stimulated cAMP production in myotubes but not in myoblasts (approximately 35% of cAMP produced by RAC alone). Turkey satellite cells (derived from biceps femoris of 12-wk-old toms) produced essentially no increased cAMP when exposed to 10 microM RAC stereoisomers. Stability of RAC was evaluated under laboratory storage and culture conditions. The RAC was stable for more than 4 mo when stored in deuterated DMSO (>98% purity) at room temperature or in aqueous solutions at -80 degrees C, as determined from sequential nuclear magnetic resonance studies. Radiolabeled RAC was incubated for 72 h in the presence of serum-containing medium, with or without C2C12 cells. Ninety-eight percent of the parent compound found in the medium at time zero was present in the medium as parent at the end of 72 h. The cellular cAMP response to RAC through beta-adrenergic receptors seems to be stereospecific. If the state of myoblasts and myotubes in vitro reflects the in vivo state, then the ractopamine effect in vivo on cellular processes (including cell division and protein and DNA accumulation) may be independent of beta-adrenergic receptors in muscle.

Adrenergic beta-Agonists↗

PubMed2000

Plasmid transfection and retroviral transduction of porcine muscle cells for cell-mediated gene transfer.

Cell-mediated gene transfer is a potential tool for studying muscle growth, but efficient genetic manipulation and implantation strategies have not been developed for pigs. The objectives of the present study were to determine methods for transient and stable incorporation of reporter genes into porcine muscle cells and to investigate their use for cell-mediated gene transfer in pigs. Porcine myoblasts and fibroblasts were isolated from muscle of 2-wk-old male pigs. Myogenic cell lines were identified using muscle-specific monoclonal antibodies, myotube fusion assays, and the presence of muscle-specific markers (MyoD and desmin). Four commercial cationic liposomes (lipofectAMINE, lipofectin, cellFECTIN, and DMRIE-C) were tested at different DNA:lipid ratios for their ability to transfect myoblasts and fibroblasts transiently with a luciferase reporter plasmid. LipofectAMINE resulted in the greatest (P < .01) transient luciferase activity for both cell types. Electroporation of cells for transient transfection resulted in less luciferase activity than cationic transfection. Stable transfections were conducted using a green fluorescence protein (GFP) reporter plasmid containing the neomycin resistance gene. LipofectAMINE transfection resulted in stable GFP expression in 1:16,000 myoblasts and 1:33,000 fibroblasts. Stable electroporation resulted in efficiencies that were significantly lower than established with cationic liposomes. Porcine cells were transduced with GFP using vesicular stomatitis virus glycoprotein G pseudotyped retrovirus and resulted in efficiencies of 1:1.2 for myoblasts and 1:1.1 for fibroblasts. These results show that cationic liposomes are superior to electroporation for transfection, but retroviral transduction produced stable reporter gene expression in > 80% of porcine muscle cells. Transduced GFP-positive cells were separated from GFP-negative cells by fluorescence-activated cell sorting and implanted into 2-wk-old male pigs. On d 4, implanted muscles were removed and subjected to immunodetection of GFP protein. Fibroblast implantation resulted in limited GFP expression within muscle, whereas myoblast implantation resulted in GFP within muscle fibers. This suggests that cell-mediated gene transfer is possible in porcine muscle and may be useful as an approach for studying muscle growth in pigs.

Animals↗