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Genetic Differentiation of Common Fox Vulpes Vulpes (Linnaeus, 1758) on the Basis of the Insulin-Like Growth Factor 1 (IGF1), Myosin-XV (MYO15A) and Paired Box Homeotic 3 (PAX3) Genes Fragments Polymorphism

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Single-nucleotide polymorphism (SNP) was analysed for selected fragments of three genes - insulin-like growth factor 1 (IGF1), myosin-XV (MYO15A) and paired box homeotic gene 3 (PAX3) - in farm and wild red foxes from two continents. The study was undertaken in order to verify whether the SNP characteristics of these genes enable farm-bred foxes to be distinguished from free-living foxes. The greatest number of changes were detected in the IGF1 gene. For each of the genes investigated specific SNP profiles characteristic only for farm foxes and only for wild foxes were noted. At the same time, specific SNP profiles were noted for wild foxes from North America and from Europe. The frequency of SNP (bases per SNP) in the gene fragments examined was 22 bp for IGF1, 34 bp for PAX3 and 56 bp for MYO15A. Single-nucleotide polymorphism is a very good molecular marker enabling characterization of nucleotide variation in the genes investigated between wild and farm individuals
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Ann. Anim. Sci., Vol. 14, No. 4 (2014) 807–819 DOI: 10.2478/aoas-2014-0052
GENETIC DIFFERENTIATION OF COMMON FOX VULPES VULPES
(LINNAEUS, 1758) ON THE BASIS OF THE INSULIN-LIKE GROWTH
FACTOR 1 (IGF1), MYOSIN-XV (MYO15A) AND PAIRED BOX
HOMEOTIC 3 (PAX3) GENES FRAGMENTS POLYMORPHISM* *
Andrzej Jakubczak, Magdalena Gryzińska, Beata Horecka, Kornel Kasperek, Katarzyna Dziadosz,
Grażyna Jeżewska-Witkowska
Department of Biological Basis of Animal Production, Faculty of Biology and Animal Breeding,
University of Life Sciences in Lublin, Akademicka 13, 20-950 Lublin, Poland
Corresponding author: andrzej.jakubczak@up.lublin.pl
Abstract
Single-nucleotide polymorphism (SNP) was analysed for selected fragments of three genes
insulin-like growth factor 1 (IGF1), myosin-XV (MYO15A) and paired box homeotic gene 3
(PAX3) – in farm and wild red foxes from two continents. The study was undertaken in order to
verify whether the SNP characteristics of these genes enable farm-bred foxes to be distinguished
from free-living foxes. The greatest number of changes were detected in the IGF1 gene. For each
of the genes investigated specic SNP proles characteristic only for farm foxes and only for wild
foxes were noted. At the same time, specic SNP proles were noted for wild foxes from North
America and from Europe. The frequency of SNP (bases per SNP) in the gene fragments examined
was 22 bp for IGF1, 34 bp for PAX3 and 56 bp for MYO15A. Single-nucleotide polymorphism is a
very good molecular marker enabling characterization of nucleotide variation in the genes inves-
tigated between wild and farm individuals.
Key words: SNP, IGF1, MYO15A, PAX3, Vulpes vulpes
Based on the latest systematics, 45 local varieties or subspecies of red fox (Vulpes
vulpes) are distinguished. Three genes were selected for the study, inuencing body
mass, hearing, and organ and tissue formation. The IGF1 gene encodes a specic
protein (insulin-like growth factor 1, somatomedin C) whose structure and function
are similar to those of insulin, included in the family of proteins that signicantly
affect growth and development (Rotwein et al., 1986). Research on mice, humans,
and canids has demonstrated that the IGF1 gene signicantly inuences body size in
*Funds of the National Centre for Research and Development (NCBiR), development project
No. 12-0140-10.
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mammals. Mice with a damaged IGF1 gene have been shown to attain a very small
size, while humans with a deletion in this gene are born with a body length consid-
erably below the norm (Sutter et al., 2007). The type of arrangement determines
the size of the individual. The arrangement of the gene together with its regulatory
sequence occurs in two forms, termed I and B. All small breeds of dog have the I
form, while large breeds have the B form. The only representatives of large breeds
with the same DNA sequence as small dogs are Rottweilers. This is because they
have other gene sequences that signicantly inuence their size (Sutter et al., 2007).
The MYO15A gene is involved in the production of a protein included in the group of
motor proteins known as myosins (Nal et al., 2007). Mutation in the MYO15A gene
leads to malfunctioning of myosin XVA and to hereditary hearing impairment in hu-
mans, mice and dogs (Kikkawa et al., 2005). The PAX3 gene plays a key role in the
formation of individual organs and tissues in the initial stages of embryonic develop-
ment, and in maintaining normal cell functions after birth. In the dog it is localized in
37q16-q17 (Krempler et al., 2000). The protein produced by PAX3 is essential during
the formation of the myotome (it induces expression of two MRFs – muscle regula-
tory factors) and acts together with the protein produced by the PAX7 gene (Lamey
et al., 2004). During embryonic development and in the muscles of the adult organ-
ism, these two proteins – PAX3 and PAX7take part in myotome formation and in
muscle growth and regeneration. Mutants without a functional PAX3 gene – splotch
mutants – are characterized by defects in the structure of the neural tube and limb
muscles (Relaix et al., 2004).
Diversity of phenotypes occurring in both wild and farm animals is an important
factor allowing for the differentiation of groups belonging to the family Canidae. The
hypothesis was that there is the SNPs differentiation in IGF1, MYO15A and PAX3
genes in the farm-bred and wild living population of common fox from Europe (Po-
land) and North America (north-eastern regions of the United States and Canada).
Although we know the localization of genes on chromosomes and their nucleotide
composition, it is not always clear how they are inherited and the way they affect
a given trait is not fully understood (e.g. complementary interaction or epistasis).
The aim of the study was to determine the nucleotide sequence of fragments of the
genes IGF1, MYO15A and PAX3 in farm and wild individuals of the Canidae family
from two continents, and identify any polymorphisms occurring in the nucleotide
sequence. In the future, this will contribute to a better understanding of the role of
these genes in heredity of morphometric traits in species of this family. The study
was undertaken in order to verify whether the SNP characteristics of these genes en-
able farm-bred foxes to be distinguished from free-living foxes.
Material and methods
Animals
The material for the study consisted of the blood of farm red foxes (Vulpes vul-
pes) from Poland (20 individuals) and raw skins of wild foxes from North America –
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north-eastern regions of the United States and Canada (20 individuals) and Poland
(20 individuals).
Genetic and data analysis
Total genomic DNA was extracted from each sample using a commercial QIA-
gen extraction kit (QIAamp DNA Blood Mini Kit or DNeasy Blood and Tissue Kit)
following the protocol provided in the QIAcube. The primers were designed using
Primer3Plus software (Rozen and Skaletsky, 2000) to amplify each region of IGF1,
MYO15A and PAX3 (http://www.bioinformatics.nl/cgi-bin/primer3plus/primer3plus.
cgi/) (Table 1).
Fragments were selected of genes responsible for different traits (body weight,
the hearing process, development of the central nervous system and melanocytes).
The genes were selected because these traits are important both physiologically and
in terms of livestock selection.
Table 1. Primers used for PCR of the three genes
Gene Forward primer (5′-3′) Reverse primer (5′-3′) Size
(bp)
IGF1 AAGTAGCCTGAGTAAGATTTGACT AGCAATCTACCAACTCCAGGACCA 305
MYO15A TTTCCACATCCACTCTCACG GAAGGGGGAGAAGCAGACTT 168
PAX3 CGACCTTGCAGCTGCTTGGGT AGTGTGGGATGCCCGCAGTG 303
The rst amplication was performed by polymerase chain reaction (PCR). The
reactions (25 μL total volume) contained 2 μl DNA (with the DNA concentration
50 ng/μl) and 1.0 U Taq polymerase (Ampli Taq Gold 360 DNA Polymerase, Ap-
plied Biosystems) in the manufacturer’s buffer, adjusted to a nal concentration of
2.5 mM MgCl2, 0.2 mM of each dNTP and 0.1 mM of each primer. PCR cycling
conditions were 95°C for 10 min; 30 cycles of 95°C for 30 s, 52°C for 60 s (IGF1
and MYO15A) or 63°C for 60 s (PAX3), 72°C for 60 s; and 72°C for 20 min. (Labcy-
cler, SensoQuest). To conrm the PCR products, gel electrophoresis was carried out
using 2% agarose gel with ethidium bromide (EtBr). The PCR product was puried
using an ExoSAP-IT kit (Affymetrix). The second amplication (sequencing PCR)
– bidirectional sequencing – was carried out according to the BigDye® Terminator
v3.1 CycleSequencing Kit (Applied Biosystems). PCR products were puried us-
ing a DyeEx Spin Kit (Qiagen) in the QIAcube. PCR products were sequenced us-
ing a 3100 Genetic Analyser (Applied Biosystems). The sequences were assembled
into consensus sequences using DNA Baser (Heracle Biosoft; http://www.DnaBaser.
com). Sequencing results were aligned using BLAST. The sequencing data were then
compared with canine reference sequences for the genes IGF1 (NCBI – ID: 610255),
MYO15A (EMBL – AJ428858) and PAX3 (NCBI ID: 488544) registered in the
NCBI database. The SNP positions of farm and wild individuals were compared
using MEGA4 software. The frequency of individual SNP proles was calculated
with the SAS statistical package and ARLEQUIN v.3.5. SNP prole was dened as
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a set of single nucleotide polymorphisms (SNPs) obtained through sequencing with
respect to each of the gene fragments investigated.
Results
The following genes were analysed: insulin-like growth factor 1 (IGF1); myo-
sin-XV (MYO15A) and paired box homeotic gene 3 (PAX3).
Substitutions
Fourteen transitions and transversions (SNPs) were observed in the IGF1 gene,
ranging from 3 to 303 bp in the region examined (intron 5, exon 6 and intron 6),
including eight transitions, at positions c.26037 (A>G), c.26144 (G>A), c.26194 and
c.26246 (C>T), c.26251 and c.26258 (A>G), c.26282 (T>C) and c.26334 (G>A),
and six transversions, at c.26032 and c.26200 (T>A), c.26043, c.26249, c.26275
(G>C) and c.26324 (C>A). In the case of the MYO15A gene fragment, three SNPs
were noted, including two transitions, at c.426 (A>G) and c.463 (G>A), and one
transversion, at c.578 (C>A). For the PAX3 gene, 9 SNPs were identied, including
6 transitions, at c.11212, c.11260, c.11395 (C>T), c.11284 (T>C), c.11437 and
c.11485 (G>A), and 4 transversions, at c.11198 (T>A), c.11273 (G>T), c.11476
(G>C) and c.11485 (G>C). Both transition and transversion occurred at position
302, which is an example of a very rare triallelic SNP locus. The frequency of oc-
currence of SNPs in the gene fragments was 1 SNP every 22 bp for IGF-1, 34 bp for
PAX3 and 56 for MYO15A.
Figure 1. Distribution of transitions and transversions among SNPs for the genes investigated
Transitions accounted for 72.8% and transversions for 27.2% of the SNPs ana-
lysed. The transition/transversion ratio (R) for the genes ranged from 2.59 to 3.0:
RIGF1=2.88; RMYO15A=3.00 and RPAX3=2.59 (Figure 1).
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Gene SNP position Frequency of SNP proles*
IGF1
c. 26032
c. 26037
c. 26043
c. 26144
c. 26194
c. 26200
c. 26246
c. 26249
c. 26251
c. 26258
c. 26275
c. 26282
c. 26324
c. 26334
All F WP WNA
SNP
proles
A A A C G C TC G A A G C A G 0.050 0.150
BA A G G C TC G A A G C C G 0.070 0.200
CTA G G C A TC A G G C C G 0.050 0.150
D T A G G C TC C A A G C C G 0.050 0.150
E T A G G C TC G A A G C C G 0.610 0.850 0.400 0.600
F T A G G C TC G A A G YC G 0.050 0.150
GTA G G C TC G A G C C C G 0.050 0.150
H T GTAT T C G G A G TC A 0.070 0.200
Genotype
frequency
AA 0.120 0.940 0.070 0.050 0.930 0.900 0.050 0.120
TT 0.880 0.070 0.070 0.950 0.050 0.070 0.880
CC 0.050 0.930 0.950 0.100 0.050 0.880 0.950
GG 0.060 0.880 0.930 0.900 0.070 0.100 0.950
TC 0.050
Allele
frequency
A0.120 0.940 0.070 0.050 0.930 0.900 0.050 0.120
T 0.880 0.070 0.070 0.950 0.050 0.095 0.880
C0.050 0.930 0.950 0.100 0.050 0.950
G0.060 0.880 0.930 0.900 0.070 0.100 0.950 0.905
Table 2. The types and frequency of SNP proles in the genes IGF1, MYO15A and PAX3 in foxes (*F – farm, WP – wild Poland, WNA – wild North America)
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Table 2 – contd.
MYO15A c. 426 c. 463 c.578 All F WP WNA
SNP
proles
A A A C 0.017 0.050
BA G A 0.050 0.150
C A G C 0.883 0.850 0.900 0.900
DARC0.017 0.050
EG G C 0.033 0.100
Genotype
frequency
AA 0.970 0.020 0.050
CC 0.950
GG 0.030 0.960
GA 0.020
Allele
frequency
A0.970 0.030 0.050
C0.950
G0.030 0.970
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Table 2 – contd.
PAX3 c. 11198 c. 11212 c. 11260 c. 11273 c. 11284 c. 11395 c. 11473 c. 11476 c. 11485 All F WP WNA
1 2 3 4 5 6 7 8 9 10 11 12 13 14
SNP
proles
A A C Y K T Y G G G 0.017 0.050
B T C C G C C G G G 0.017 0.050
CTC C G TC A G G 0.017 0.050
D T C C G TC G G A 0.017 0.050
E T C C G TC G G G 0.100 0.200 0.100
F T C C G T T G G G 0.033 0.050 0.050
GTC C G T Y G G G 0.033 0.100
H T C C G YC G G G 0.100 0.150 0.150
I T C C K Y Y G G G 0.017 0.050
JTCTG C C G G G 0.017 0.050
K T CTGT T G G G 0.017 0.050
L T CTGT Y G C C 0.017 0.050
MTCT K T C A G G 0.017 0.050
N T CT K T C G G G 0.100 0.250 0.050
O T CT K T Y G G G 0.017 0.050
P T CT K Y Y G G G 0.062 0.200
R T CT T T C G G G 0.017 0.050
S T CT T Y C G G G 0.050 0.050 0.100
T T CYGTC G G G 0.017 0.050
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Table 2 – contd.
1 2 3 4 5 6 7 8 9 10 11 12 13 14
UTCYGYC G G G 0.017 0.050
W T CY K T C G G G 0.017 0.050
X T CY K Y C G G G 0.017 0.050
Y T CY K Y Y G G G 0.017 0.050
ZTCY T T Y G G G 0.050 0.150
A1 T CY T Y C G G G 0.017 0.050
B1 T CY T Y Y G G G 0.033 0.100
C1 T T C G TC G G G 0.033 0.100
D1 T Y C G TC G G G 0.033 0.100
E1 T Y C G T Y G G G 0.033 0.100
F1 T Y Y GTC G G G 0.017 0.050
G1 T Y Y GYC G G G 0.017 0.050
H1 T Y Y K Y C G G G 0.017 0.050
Genotype
frequency
AA 0.020 0.030 0.020
TT 0.980 0.030 0.320 0.170 0.600 0.050
CC 0.850 0.430 0.030 0.650 0.020 0.020
GG 0.530 0.970 0.980 0.960
TC 0.120 0.250 0.370 0.300
GT 0.300
Allele
frequency
A0.020 0.030 0.020
T 0.980 0.090 0.445 0.320 0.785 0.050
C0.910 0.555 0.215 0.650 0.020 0.020
G0.680 0.300 0.970 0.980 0.960
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IGF1
The IGF1 gene fragment studied in the red foxes consists of 305 bp. The statisti-
cal analysis showed 14 mutations, allowing 8 SNP proles (A-H) to be distinguished.
Prole F was found exclusively in farm animals, while proles A, B, C, D, G and H
were present in wild animals. B and H were found in North American wild foxes, and
the remaining prole in Polish wild foxes, which may have been due to the animals’
different living conditions and adaptation to climate. Only prole E was present in
all of the populations, which suggests that this set of SNP is inherited in both farm
and wild animals (Table 2).
MYO15A
The MYO15A gene fragment analysed was 168 bp in length. Three SNPs were
noted (two mutations in wild foxes and one in farm animals) and ve SNP proles
(A-E). Proles A and D were present only in foxes living in their natural habitat in
Poland. Prole B was found only in farm animals. Only prole C was noted in all
individuals and had the highest frequency. Prole E occurred with relatively low
frequency, and only in North American wild foxes.
PAX3
The PAX3 gene fragment analysed in the red fox was 303 bp in length. The statis-
tical analysis showed 32 different SNP proles, caused by 9 single-nucleotide poly-
morphisms. There were 8 characteristic SNP proles noted for the farm foxes and
2 for the wild foxes (North American and Polish). At the same time, 12 SNP proles
were noted in the North American wild foxes (with a frequency of 5% to 10%), and
7 for Polish wild foxes (with a frequency of 5% to 20%). No SNP prole was noted
that was characteristic of all three groups of foxes (Table 2).
Frequency of genotypes and alleles for specic SNP are shown in Table 2, and the
gene diversity and nucleotide diversity between any two DNA sequences are shown
in Table 3.
Table 3. Gene diversity and nucleotide diversity of the genes IGF1, MYO15A and PAX3 in foxes
(*F – farm, WP – wild Poland, WNA – wild North America)
Gene Gene diversity in population* Nucleotide diversity (π)
(average over loci) in population*
FWP WNA F WP WNA
IGF1 0.2684 0.7895 0.5895 0.019173 0.178195 0.192481
MYO15A 0.1895 0.1947 0.1895 0.089474 0.031579 0.063158
PAX3 0.9316 0.8947 0.9737 0.049123 0.054386 0.093567
Discussion
The completion of the dog genome sequencing project made it possible to nd
SNPs in the genomes of animals of the Canidae family. Sacks and Louie (2008) used
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40 different primer pairs designed using random fragments of the dog genome to
sequence 80–88% of loci in these fragments in the coyote (Canis latrans), the gray
fox (Urocyon cinereoargenteus) and the red fox (Vulpes vulpes). In order to inves-
tigate the genetic basis for size variation in canids, Sutter et al. (2007) searched for
SNPs in a region composed of 15 million base pairs. They discovered 302 SNPs and
34 indels in large and small Portuguese Water Dogs (PWD). The IGF1 gene was
shown to have a signicant inuence on individual size. Haplotypes B and I were
identied. Dogs that were homozygous with haplotype B have a smaller mean body
size than homozygotes with haplotype I, while heterozygous dogs are medium-sized.
In a study of 122 SNPs spanning chromosome 15 in dogs representing 14 small and
9 giant breeds, a decrease in heterozygosity was observed in the small breeds, which
may be due to selective breeding resulting in smaller and smaller dogs. Research
concerning SNPs in the IGF1 gene has also been carried out in other animal species.
SNPs have been detected in the coding sequence of IGF1 among ve breeds of pig
(Berkshire, Duroc, Landrace, Yorkshire and Korea Native Pig) (Niu et al., 2013), and
SNPs have been found to be associated with growth, development, and fertility in
cattle (Holstein-Friesian) (Mullen et al., 2011).
Mutations in the MYO15A gene cause congenital deafness in humans (DFNB3)
and in mice (Shaker-2). In mice, the hair cells affected by Shaker-2 deafness are ar-
ranged normally, but their length is markedly reduced. Probst et al. (1998) compared
healthy and Shaker-2 individuals and determined that the cause of the hearing loss
was a mutation in the MYO15A gene. The G>A substitution causes changes in the
motor domain of the protein involving a Cyst/Tyr substitution, leading to shortening
of the stereocilia. Rak et al. (2002) used human cDNA to design primers specic
for dogs, which were then used for RH mapping. PCR yielded a product of 201 bp
containing exclusively canine DNA. In addition, FISH (uorescence in situ hybridi-
zation) was carried out in order to map the position of MYO15A on chromosome 5 in
dogs (CFA5). This made it possible to ascertain that mutations in the MYO15A gene
in dogs can cause hereditary deafness. The present study on red foxes demonstrated
the presence of three SNP-type polymorphisms in the MYO15A gene. In Polish wild
foxes two SNP proles were distinguished – A and D, while only North American
wild foxes had SNP prole E.
Due to the importance of the processes involving proteins encoded by PAX3, mu-
tations in the genes encoding them can lead to very serious disturbances in embryotic
development, and in extreme cases can even be lethal. There are currently no publi-
cations concerning mutations of the PAX3 gene in animals of the Canidae family, but
we have identied 9 polymorphisms of the SNP type in individuals from this family,
allowing 32 different SNP proles to be distinguished. The greatest number of ani-
mals tested had prole H, which occurred in the farm and the North American wild
foxes, and prole N, which was characteristic of Polish and North American wild
foxes. At the same time, SNP proles were identied among wild foxes that were
characteristic only for North American animals and only for European ones. The
most likely cause of the development of separate SNP proles was the geographical
barrier and divergent evolutionary paths of the species Vulpes vulpes in Europe and
North America (Aubry et al., 2009; Kutschera et al., 2013).
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There is greater probability of nucleotide transitions than transversions, as the
substitution takes place between structurally similar compounds, while transversions
involve compounds of different structure. Another factor during substitution that
should be considered is the varied rate of evolution between particular sites within
codons, resulting from degeneration of the genetic code (Wondji et al., 2007). The
very high proportion of C↔T transitions (Figure 1) may result from C-methylation
in the CpG dinucleotide (Gryzińska et al., 2013 a, b). 5mC is spontaneously deami-
nated to T, causing the formation of a mismatched T:G pair and often a C:G→T:A
transition (Holliday and Grieg, 1993). Although in humans TDG (thymine-DNA gly-
cosylase) preferentially removes T from the T:G mismatch, the frequency of deami-
nation exceeds its capacity, which leads to suppression of the amount of CpG. The
transition/transversion ratio is similar to that obtained for Drosophila and humans
(Brookes, 1999; Moriyama and Powell, 1996). The frequency of occurrence of one
SNP was from 22 to 56 bp – lower than in Anopheles gambiae (Morlais et al., 2004)
and in humans (Aquadro et al., 2001).
Single-nucleotide polymorphism is a very good molecular marker enabling char-
acterization of variation between wild and farm individuals. The conrmation of
genetic differentiation between wild and farm-bred animals of the Canidae family
are reports by Bugno-Poniewierska et al. using the FISH technique (Bugno-Poniew-
ierska et al., 2012, 2013). Variation studies of different species of fur animals of the
family Canidae, free-living and farm-bred, have been and are carried out on sev-
eral levels: diversity of morphological, physiological parameters, phenotypic traits,
as well as at the molecular and cytogenetic level (Gugołek et al., 2012; Jeżewska-
Witkowska et al., 2012; Ślaska et al., 2010). However, to date in world literature
there are no papers considering the above-mentioned differences using sequence of
nuclear gene fragments: insulin-like growth factor 1 (IGF1), myosin-XV (MYO15A)
and paired box homeotic 3 (PAX3).
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Genetic differentiation of farmed and wild foxes 819
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Received: 29 X 2013
Accepted: 30 V 2014
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... It is known from literature data that fibroblast growth factor (FGF-2) is able to stimulate mitosis of human bone marrow cells (Ratajczak et al. 1996) and support proteins associated with pluripotency in cat embryonic cells (Zhou et al. 2019). In turn, insulin-like growth factor (IGF-1) is a weak mitogen for most human cells cultured (Niedźwiedzka 2000;Jakubczak et al. 2014). The growth hormone (rhGH) promotes cell proliferation, in particular, there is evidence of its positive effect on human mesenchymal stem cells (Li and Wang 2008;Waters and Brooks 2012). ...
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Chapter
Designing PCR and sequencing primers are essential activities for molecular biologists around the world. This chapter assumes acquaintance with the principles and practice of PCR, as outlined in, for example, refs. 1–4.