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UNIVERSIDADE FEDERAL DE PELOTAS Programa de Pós-Graduação em Biotecnologia
TESE
Nanobiotecnologia aplicada à transgênese animal
Vinicius Farias Campos
Pelotas, 2011.
VINICIUS FARIAS CAMPOS
Nanobiotecnologia aplicada à transgênese animal
Tese apresentada ao Programa de Pós-Graduação em Biotecnologia da Universidade Federal de Pelotas, como requisito parcial à obtenção do título de Doutor em Ciências (área do conhecimento: Biotecnologia).
Orientador: Prof. Tiago Collares, Dr. Co-orientadores: Profa. Fabiana Kömmling Seixas, Dra. Prof. João Carlos Deschamps, PhD.
Pelotas, 2011.
Dados de catalogação na fonte: Maria Beatriz Vaghetti Vieira – CRB 10/1032 Biblioteca de Ciência & Tecnologia - UFPel
C198n Campos, Vinicius Farias
Nanobiotecnologia aplicada à transgênese animal / Vinicius Farias Campos. – 75f. : tab. – Tese (Doutorado). Programa de Pós-Graduação em Biotecnologia. Universidade Federal de Pelotas. Centro de Desenvolvimento Tecnológico, 2011. – Orientador Tiago Collares; co-orientador Fabiana Kömmling Seixas, João Carlos Deschamps.
1.Biotecnologia. 2.SMGT. 3.Nanopolímero catiônico.
4.Bovinos. 5.Nanotubos. 6.Transferência gênica. 7.Sêmen. 8.Nanobiotecnologia. I.Collares, Tiago. II.Seixas, Fabiana Kömmling. III.Deschamps, João Carlos. IV.Título.
CDD: 636.2
Banca examinadora:
Prof. Dr. Tiago Collares, Universidade Federal de Pelotas Prof. Dr. João Carlos Deschamps, Universidade Federal de Pelotas Profª. Dra. Fabiana Kömmling Seixas, Universidade Federal de Pelotas Profª. Dra. Marta Gonçalves Amaral, Universidade Federal de Pelotas
Dedico esta tese aos meus pais.
AGRADECIMENTOS
Aos meus pais Noli e Clair pelo carinho, por toda a sua dedicação na minha
formação, pela força e incentivo nas horas mais difíceis, apesar da nossa grande
distância e saudades.
Ao meu Irmão, minha cunhada e meus sobrinhos pelo carinho e alegria
despendida.
À minha esposa Heren, pelo companheirismo e incentivo.
Ao meu orientador e meu grande amigo Prof. Tiago Collares por sua
dedicação na minha formação, paciência, sinceridade e pelos ensinamentos.
Ao meu co-orientador e amigo Prof. Deschamps, por ter acreditado em mim e
pelos seus ensinamentos.
À minha co-orientadora e amiga Profa. Fabiana, por toda sua dedicação em
minha formação e ensinamentos.
Aos meus amigos e parceiros nos experimentos da tese Priscila, Eliza e
Gabriel.
Aos colegas do grupo de pesquisa em Oncologia Celular e Molecular, Helena,
Fernanda Nedel, Felipe, Virgínia, Samuel, Fernando, Fernanda Rodrigues, Eduarda,
Ruan, Ludmila, Marrí, Karine, Cristian, Stéphanie, Suélen, Carol, Emily, Cris e
Vanessa pela amizade e colaboração no dia a dia.
Aos amigos e colegas do Lab. 10, Leonardo, Marta, Cláudia e Thais, por toda
força quando necessário e pelos momentos agradáveis.
Aos professores do Programa de Pós-Graduação em Biotecnologia pelos
ensinamentos.
Aos amigos e colegas do Centro de Biotecnologia pelo auxílio nas demais
atividades e convivência agradável.
A todos que colaboraram de alguma forma para a execução deste trabalho.
À Universidade Federal de Pelotas e ao Programa de Pós-Graduação em
Biotecnologia pela oportunidade de realizar um qualificado curso de doutorado.
A CAPES pela bolsa de estudos.
A todos muito OBRIGADO!!!
I get by with a little help from my friends
I get high with a little help from my friends
Gonna try with a little help from my friends
John Lennon e Paul McCartney
RESUMO
CAMPOS, Vinicius Farias. Nanobiotecnologia aplicada à transgênese animal. 2011. 75f. Tese (Doutorado) – Programa de Pós-Graduação em Biotecnologia, Universidade Federal de Pelotas, Pelotas. A nanobiotecnologia tem proporcionado novos avanços científicos e tecnológicos em
diversas áreas do conhecimento tornando-se assim área de pesquisa prioritária em
países desenvolvidos e em desenvolvimento. A transferência gênica mediada por
espermatozóides (SMGT) poderá se tornar a técnica mais simples, eficiente e com
melhor custo-benefício para a geração de animais transgênicos. O desenvolvimento
de nanocompósitos capazes de carrear o DNA exógeno para o interior de células
com maior eficiência permite que técnicas como a SMGT sejam aperfeiçoadas. A
NanoSMGT é uma técnica utilizada para a geração de animais transgênicos onde a
nanotecnologia é utilizada para incrementar a habilidade dos espermatozóides em
capurar o DNA exógeno. O objetivo do presente trabalho foi de verificar se
nanopolímero catiônico ou nanotubos de haloisita são capazes de transfectar o DNA
exógeno para o interior de espermatozóides bovinos sexados e não sexados e em
seguida verificar se estes espermatozóides transfectados são capazes de gerar
embriões bovinos transgênicos. Utilizando PCR em tempo real, verificou-se que o
nanopolímero catiônico é capaz de introduzir o DNA exógeno em espermatozóides
bovinos sexados e não sexados sem danos para a motilidade e viabilidade
espermática. Também foi demonstrado pela primeira vez que o nanopolímero
catiônico ou os nanotubos de haloisita são capazes de incrementar tanto a
transfecção de DNA em espermatozóides como a transmissão do transgene para
embriões bovinos produzidos in vitro. Estes resultados demonstram que a
NanoSMGT pode ser uma técnica viável para a produção de embriões bovinos
transgênicos.
Palavras-chave: SMGT, bovinos, transfecção, nanobiotecnologia, nanopolímero
catiônico, nanotubos.
ABSTRACT
CAMPOS, Vinicius Farias. Nanobiotecnologia aplicada à transgênese animal. 2011. 75f. Tese (Doutorado) – Programa de Pós-Graduação em Biotecnologia, Universidade Federal de Pelotas, Pelotas.
Nanobiotechnology has provided new scientific and technological knowledge in
distinct areas making it a priority area of research in developed and developing
countries. The sperm-mediated gene transfer (SMGT) technique may become more
simple, efficient and cost-effective technique for the generation of transgenic animals.
The development of nanocomposites able to carry foreign DNA into the nucleus of
cells with greater efficiency allows techniques such as SMGT be improved. The
NanoSMGT is a technique used to generate transgenic animals in which
nanotechnology is used to enhance the ability of sperm to capture exogenous DNA.
The objective of this study was to determine whether cationic nanopolymer or
halloysite clay nanotubes are able to transfect the exogenous DNA to unsorted and
sex-sorted bovine sperm then evaluate whether these sperm are able to transmit
transgene to in vitro fertilized bovine embryos. Using real-time PCR, we found that
the cationic nanopolymer is capable of introducing exogenous DNA into unsorted and
sex-sorted bovine sperm without negative effects to sperm motility and viability. Was
also demonstrated for the first time that cationic nanopolymer or halloysite clay
nanotubes are able to increase both the sperm DNA transfection of as the
transmission of the transgene to bovine embryos produced in vitro. These results
demonstrate that NanoSMGT can be a viable technique for producing transgenic
bovine embryos.
Keywords: SMGT, cattle, transfection, nanobiotechnology, cationic nanopolymer,
nanotubes.
LISTA DE ABREVIATURAS E SIGLAS
ANOVA – Análise de variância
BSA – Albumina sérica bovina
CMV – Citomegalovírus
COC – Complexo cumulus-oócito
DMSO – Dimetilsulfóxido
DNA – Ácido desoxirribonucléico
EGFP – Proteína verde fluorescente
FSH – Hormônio folículo-estimulante
HCN – Nanotubos de haloisita
ICSI – Injeção intracitoplasmática de espermatozóides
IVF – Fertilização in vitro
IVM – Maturação in vitro
NanoSMGT – Nanotransferência gênica mediada por espermatozóides
NTC – Controle sem DNA
PCR – Reação em cadeia da polimerase
qPCR – Reação em Cadeia da Polimerase quantitativa
SOF – Fluído sintético do oviducto
GH – Hormônio do crescimento
SUMÁRIO
1. INTRODUÇÃO GERAL ............................................................................ 11
1.1. Animais transgênicos ......................................................................... 11
1.2. Técnicas para a geração de animais transgênicos ............................ 13
1.3. Transferência gênica mediada por espermatozóides ........................ 14
1.4. Nanobiotecnologia e a transferência gênica mediada por espermatozóides ...................................................................................... 16
2. ARTIGO 1 ................................................................................................. 19
Abstract .................................................................................................... 21
1. Introduction ........................................................................................... 22
2. Materials and methods ......................................................................... 23
3. Results .................................................................................................. 27
4. Discussion ............................................................................................ 28
References ............................................................................................... 31
Figure captions ......................................................................................... 36
3. ARTIGO 2 ................................................................................................. 39
Abstract .................................................................................................... 41
1. Introduction ........................................................................................... 42
2. Materials and methods ......................................................................... 43
3. Results .................................................................................................. 52
4. Discussion ............................................................................................ 54
References ............................................................................................... 58
Figure Captions ........................................................................................ 64
4. CONCLUSÃO ........................................................................................... 67
5. REFERÊNCIAS ........................................................................................ 68
11
1
2
3
4
5
6
7
1. INTRODUÇÃO GERAL 8
9
1.1. Animais transgênicos 10
11
Na moderna biotecnologia o ano de 1982 foi marcado por dois 12
importantes eventos. Primeiro, neste ano deu-se início a comercialização da 13
insulina recombinante, produzida em bactérias, sendo este o primeiro produto 14
oriundo da engenharia genética utilizado na terapia de seres humanos. Ainda, 15
em dezembro deste ano, PALMITER et al., (1982), demonstraram pela primeira 16
vez a geração de um animal transgênico, através da microinjeção pronuclear 17
em embriões zigóticos de um fragmento de DNA contendo o promotor da 18
metalotioneína e o gene do hormônio do crescimento (GH) de ratos. Estes 19
animais geneticamente modificados apresentavam um crescimento muito 20
superior em ralação aos animais não-transgênicos. A partir daquele ano, os 21
animais transgênicos vêm sendo desenvolvidos para diversos propósitos, entre 22
eles a produção de proteínas recombinantes (LILLICO et al., 2007), o 23
melhoramento genético animal (MILAZZOTTO et al., 2010) e a geração de 24
modelos para estudos de doenças, principalmente para o câncer (WALRATH et 25
al., 2010). 26
A transgênese tornou-se uma importante ferramenta para o campo da 27
biologia e atualmente pelo menos 90% dos animais geneticamente modificados 28
são gerados para os estudos básicos. Estudos recentes têm demonstrado que 29
os animais transgênicos domésticos são mais apropriados para serem usados 30
como modelos para o estudo de doenças humanas (KUES; NIEMANN, 2011). 31
Um modelo de suíno transgênico para uma doença humana rara nos olhos 32
chamada retinitis pigmematosa foi desenvolvido recentemente (JAKOBSEN et 33
al., 2011). Embora os animais domésticos venham sendo desenvolvidos para 34
12
serem usados como modelos de doenças, os camundongos transgênicos ainda 1
são os modelos mais utilizados nesta área da transgênese, devido ao seu 2
baixo custo, rápida reprodução e fácil manipulação genética. 3
Por outro lado, os animais domésticos vêm sendo utilizados na 4
transgênese em duas principais frentes, na agricultura, contemplando o 5
melhoramento genético animal, e na saúde, para a produção de proteínas 6
recombinantes para terapia de doenças humanas. Suínos transgênicos com 7
superexpressão da α-lactoalbumina na glândula mamária induziram um maior 8
nível de lactose e produção de leite, aumentando, conseqüentemente a 9
sobrevivência e o desenvolvimento dos leitões (WHEELER; BLECK; 10
DONOVAN, 2001). Este aumento da sobrevivência dos leitões ao desmame 11
proporcionaria significativos benefícios comerciais para o produtor e à saude 12
dos animais. A proteína lisostafina confere resistência específica contra a 13
mastite causada por Staphylococcus aureus. Vacas transgênicas resistentes a 14
esta infecção foram produzidas por expressar o gene da lisostafina na glândula 15
mamária (WALL et al., 2005). Estes resultados demonstram a viabilidade de 16
produzir alterações significativas na composição do leite através da aplicação 17
de uma estratégia transgênica adequada. 18
Apesar das aplicações no melhoramento genético, os animais 19
domésticos transgênicos vêm sendo mais explorados para a produção de 20
proteínas recombinantes no conhecido gene pharming, que implica na 21
produção de proteínas recombinantes no leite de vacas, cabras ou ovelhas 22
(HOUDEBINE, 2009). A glândula mamária têm sido o fluido eleito com maior 23
freqüência para expressão de proteínas recombinantes devidos aos altos níveis 24
de proteína que podem ser produzidos e aos métodos para extração e 25
purificação já elucidados (KUES; NIEMANN, 2011). Vários produtos derivados 26
da glândula mamária de cabras e ovelhas transgênicas evoluíram para estágio 27
avançado de testes clínicos. Os ensaios clínicos de fase III para antitrombina III 28
(ATIII) (ATryn da GTC-Biotherapeutics, EUA), produzido na glândula mamária 29
de cabras transgênicas foi concluído e o produto recombinante foi aprovado 30
como droga terapêutica pela Agência Europeia de Medicamentos (EMA), em 31
agosto de 2006 e nos EUA pelo FDA em fevereiro de 2009. Esta proteína é o 32
primeiro produto a partir de um animal transgênico de produção a ser aceito 33
como uma droga totalmente registrada. O ATryn está registrado para o 34
13
tratamento de pacientes submetidos a procedimentos de circulação 1
extracorpórea que são resistentes à heparina. A GTC-Biotherapeutics também 2
já expressou pelo menos outras onze proteínas recombinantes na glândula 3
mamária de cabras transgênicas. 4
Anticorpos monoclonais também estão sendo produzidos na glândula 5
mamária de cabras transgênicas bem como em vacas transgênicas clonadas 6
que foram criadas para que produzam um anticorpo recombinante bi-específico 7
em seu sangue (GROSSE-HOVEST et al., 2004). Um desenvolvimento 8
interessante é a geração animais trans-cromossômicos portadores de um loci 9
com a seqüência completa da cadeia pesada e leve da imunoglobulina humana 10
em um cromossomo humano artificial (HAC). Este sistema é um passo 11
significativo na produção de anticorpos policlonais para terapêutica humana 12
(KUROIWA et al., 2009). Estudos de acompanhamento mostraram que o HAC 13
manteve-se em clones fundadores durante vários anos (ROBL et al., 2007). 14
Entretanto, ainda não se conhece como os HACs se manterão durante as 15
divisões celulares meióticas das células. 16
17
1.2. Técnicas para a geração de animais transgênicos 18
19
A produção de animais transgênicos é um trabalho intensivo e de alto 20
custo e depende de técnicas avançadas de biologia molecular, cultura de 21
células, biologia reprodutiva e bioquímica. O primeiro método de transferência 22
gênica bem sucedido em camundongos foi baseada na microinjeção de DNA 23
exógeno em pronúcleos zigóticos (GORDON et al., 1980; PALMITER et al., 24
1982). A injeção pronuclear de DNA também foi usada para produzir o primeiro 25
animal transgênico de produção a mais de 20 anos atrás (HAMMER et al., 26
1985). Apesar da ineficiência inerente da tecnologia de microinjeção, um amplo 27
espectro de organismos geneticamente modificados tem sido gerados para 28
aplicações na agricultura e na biomedicina. 29
Várias alternativas para a microinjeção de DNA pronuclear têm sido 30
desenvolvidas nos últimos anos para melhorar a eficiência e reduzir o custo da 31
geração de animais transgênicos. Estes incluem a transferência gênica 32
mediada por espermatozóides (CHANG et al., 2002; COLLARES et al., 2010), 33
a injeção intracitoplasmática de espermatozóides (ICSI) carregando DNA 34
14
exógeno (GARCIA-VAZQUEZ et al., 2010; PEREYRA-BONNET et al., 2011), a 1
injeção ou infecção de oócitos e/ou embriões por vetores retro e lentivirais 2
(HOFMANN et al., 2004) e o uso de transferência nuclear de células somáticas 3
(SCNT) (YANG et al., 2011). Até o presente momento, a SCNT tem sido bem 4
sucedida em mais de 16 espécies de mamíferos. Entretanto, a taxa de sucesso 5
desta técnica ainda é baixa, sendo geralmente, entre 1-3% a taxa de 6
transgênese dos embriões transferidos (MENDICINO et al., 2011). Os bovinos 7
parecem ser uma exceção a esta regra onde níveis 15-20% podem ser 8
alcançados (KUES; NIEMANN, 2011). 9
Das técnicas descritas acima, a transferência gênica mediada por 10
espermatozóides (SMGT) poderá tornar-se o método com melhor custo-11
benefício para a geração de animais transgênicos, o que aumentará 12
significantemente a aplicação dos animais transgênicos na produção comercial 13
e na pesquisa biomédica (KANG et al., 2008; CAMPOS et al., 2011a). 14
15
1.3. Transferência gênica mediada por espermatozóides 16
17
No início dos anos 70, BRACKETT et al. (1971) demonstraram que 18
moléculas de DNA exógeno poderiam ser incorporadas em um espermatozóide 19
de mamífero. Quase vinte anos depois, foi demonstrada a geração de 20
camundongos transgênicos após a fertilização de oócitos que foram 21
simplesmente incubados com o DNA alvo (LAVITRANO et al., 1989). Em 22
contraste com metodologias atuais de transferência de genes em animais de 23
produção como microinjeção de DNA em pró-núcleos fertilizados ou 24
transferência nuclear utilizando células de doadores transgênicos, que são de 25
trabalho, tempo ou custo intensivo (KUES; NIEMANN, 2011), a transferência 26
gênica mediada por espermatozóides (SMGT), provou ser reprodutível e seria 27
a mais simples e mais rápida maneira de produzir animais transgênicos 28
(LAVITRANO et al., 2006). Embora estudos anteriores tenham resultado na 29
criação de animais transgênicos em várias espécies, inclusive animais 30
marinhos invertebrados e peixes (LANES; SAMPAIO; MARINS, 2009; 31
COLLARES et al., 2010), aves (NAKANISHI; IRITANI, 1993; HAREL-32
MARKOWITZ et al., 2009), camundongos (MAIONE et al., 1998), porcos 33
(GARCIA-VAZQUEZ et al., 2010) e bovinos (SHEMESH et al., 2000; 34
15
HOELKER et al., 2007), a eficácia ainda é baixa devido a uma série de 1
problemas não resolvidos. 2
Um dos principais problemas da SMGT está relacionado com a baixa 3
incorporação de DNA exógeno pelo espermatozóide. Mesmo que a absorção 4
de DNA ocorra espontaneamente (ANZAR; BUHR, 2006), esta etapa é crítica, 5
porque o aumento captação DNA pelos espermatozóides, geralmente leva a 6
uma diminuição da motilidade e conseqüentemente da capacidade de 7
fertilização (FEITOSA et al., 2009). Portanto, novas estratégias como 8
eletroporação de espermatozóides (RIETH; POTHIER; SIRARD, 2000), 9
tratamento de espermatozóides com Triton-X (GARCIA-VAZQUEZ et al., 2009) 10
e use o de reagentes químicos de transfecção como lipossomas (HAREL-11
MARKOWITZ et al., 2009) ou DMSO (SHEN et al., 2006) foram desenvolvidos 12
para aumentar absorção de DNA exógeno em espermatozóides. No entanto, 13
mesmo que a freqüência de incorporação de DNA em espermatozóides seja 14
alta, a proporção de filhotes transgênicos permanece extremamente variável 15
(COLLARES et al., 2010). Além disso, a ação de enzimas DNases, que 16
degradam o DNA exógeno tanto no plasma seminal quanto no interior do 17
espermatozóide, pode influenciar significativamente de maneira negativa a 18
transferência do transgene para o oócito (LANES; SAMPAIO; MARINS, 2009; 19
COLLARES et al., 2010; CAMPOS et al., 2011a). Assim, mesmo que o 20
transgene seja transmitido com sucesso para o oócito, o gene repórter 21
raramente é expresso em um número expressivo de embriões e mesmo que os 22
genes exógenos sejam expressos pelo embrião transgênico pode ainda, haver 23
uma falta de integração ao genoma (SPADAFORA, 2007). Recentemente, 24
novos agentes transfectantes como nanocompósitos, tem sido usados para 25
proteger o DNA exógeno da ação destas DNases e assim incrementar o 26
processo de transfecção de DNA para as células espermáticas (CAMPOS et 27
al., 2011b). Além disso, novas estratégias como a transfecção de DNA para 28
espermatozóides sexados trazem a possibilidade de gerar animais 29
transgênicos com sexo predefinido através da SMGT (CECCO et al., 2010; 30
CAMPOS et al., 2011b). 31
Embora a principal aplicação da SMGT seja para a produção de animais 32
transgênicos, estão previstas aplicações na área humana, onde esta técnica 33
poderia ser usada para a transferência de genes na terapia gênica de 34
16
espermatozóides e testículos (PARRINGTON; COWARD; GADEA, 2011). 1
Entretanto, uma série de problemas ainda precisa ser resolvida, os 2
procedimentos para SMGT necessitam de grandes melhorias e novas 3
estratégias precisam ser desenvolvidas. 4
5
1.4. Nanobiotecnologia e a transferência gênica mediada por espermatozóides 6
7
A maioria dos nanocompósitos foram produzidos principalmente para 8
aplicações nas áreas de engenharia e de materiais, mas as tendências 9
recentes trouxeram essas ferramentas para as áreas da medicina e 10
biotecnologia. Desde o final dos anos 1970, as nanopartículas e 11
nanocompósitos têm sido utilizados para o conhecido drug delivery (KREUTER; 12
TAUBER; ILLI, 1979). Recentemente, os nanocompósitos como nanapartículas 13
e nanotubos vêm sendo utilizados como estratégia na terapia gênica devido às 14
suas propriedades que incrementam a transfecção de DNA para as células alvo 15
(DE LA FUENTE et al., 2006). A lipofecçao transporta os plasmídeos através 16
da membrana plasmática por endocitose, onde o seu tráfico intracelular tem 17
que continuar por uma via endossomal ou lisossomal. Isto faz com que a 18
maioria destas moléculas de DNA exógeno sejam degradadas por nucleases. 19
Este é também o caso quando o DNA exógeno é transfectado na ausência de 20
transfectantes – frequentemente utilizado na SMGT – onde os transgenes 21
internalizados são rapidamente degradados por DNases presente no esperma 22
(KANG et al., 2008; CAMPOS et al., 2011a). Além disso, DNases presentes no 23
líquido seminal também podem degradar moléculas de DNA exógeno. KIM et 24
al. (2010) demonstraram que, quando lipossomos foram utilizados, o 25
tratamento com DNase I reduziu significativamente a taxa de ligação a DNA 26
exógeno aos espermatozóides, e que quando nanopartículas magnéticas foram 27
utilizadas a redução na ligação foi significativamente menor. Foi demonstrado 28
que as nanopartículas magnéticas que estão vinculados ao DNA exógeno 29
podem se localizar tanto na parte interna da membrana plasmática ou no 30
interior da membrana plasmática, enquanto a maioria dos lipossomas localiza-31
se no exterior da membrana plasmática. 32
Vários nanopolímeros catiônicos ou dendrímeros têm sido utilizados 33
para incrementar a eficiência de transfecção tanto in vivo quanto in vitro (YAO 34
17
et al., 2009). Geralmente, esses agentes têm abundantes aminas primárias que 1
prontamente forma poliplexos com o DNA carregado negativamente e, 2
posteriormente, penetram para o ambiente endossomal, facilitando a liberação 3
de DNA para o citosol (FISCHER et al., 1999). Além disso, estudos anteriores 4
demonstraram que os nanotubos têm a capacidade de proteger o DNA 5
exógeno de clivagem enzimática por DNases. Os mecanismos de proteção 6
ainda não são bem compreendidos (WU et al., 2008), no entanto, a hipótese de 7
que nanotubos de sílica podem atuar como uma barreira física que protege os 8
materiais de danos na base de que nucleases celulares/proteínas não podem 9
ter acesso físico ao DNA (CHEN et al., 2005). 10
Tem sido sugerido que os nanotubos de haloisita (HCNs) podem ser 11
utilizados para a transfecção de DNA em células eucarióticas. HCNs são um 12
nanomaterial formado naturalmente com depósitos em vários países como os 13
EUA e no Brasil (LEVIS; DEASY, 2002). Os nanotubos de haloisita são 14
biocompatíveis e espontaneamente incorporados pelas células. Eles possuem 15
um lúmen interno cuja dimensão é compatível com muitas macromoléculas e 16
proteínas, permitindo a entrada e liberação de uma gama de agentes ativos 17
(VERGARO et al., 2010). Estas características fazem deste nanomaterial um 18
agente de transfecção potencialmente importante para o uso em SMGT. 19
No entanto, estudos utilizando nanopartículas para a transfecção de 20
células espermáticas ainda estão em um estágio inicial. Se o potencial da 21
nanobiotecnologia é fornecer produtos para as células, porque não utilizar este 22
potencial para a transfecção de DNA em espermatozóides? 23
Com base no exposto acima, o presente trabalho teve por objetivo 24
utilizar nanocompósitos como nanopolímeros e nanotubos para transfectar 25
DNA exógeno em espermatozóides bovinos e avaliar a eficiência destes 26
nanocompósitos em transmitir estas moléculas de DNA para embriões bovinos 27
através da técnica de NanoSMGT. 28
Os dados gerados nesta tese estão apresentados na forma de artigos 29
científicos. O primeiro artigo demonstra a transfecção de DNA exógeno em 30
espermatozóides bovinos sexados utilizando um nanopolímero catiônico. Este 31
trabalho foi publicado no periódico Theriogenology em 2011. 32
O segundo artigo demonstra que a técnica de NanoSMGT incrementa a 33
transmissão do transgene para embriões bovinos quando um nanopolímero 34
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catiônico ou nanotubos de haloisita são utilizados para a transfecçao de DNA 1
em substituição de lipossomos ou DNA puro. Este trabalho foi aceito para 2
publicação também no periódico Theriogenology. 3
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2. ARTIGO 1 8
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NanoSMGT: transfection of exogenous DNA on sex-sorted bovine sperm 13
using nanopolymer 14
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(Publicado no periódico Theriogenology, v.75, p.1476-1481, 2011.) 16
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NanoSMGT: transfection of exogenous DNA on sex-sorted bovine sperm 1
using nanopolymer 2
3
Vinicius F. Campos1, Eliza R. Komninou2, Gabriel Urtiaga1, Priscila M. de 4
Leon1, Fabiana K. Seixas1, Odir A. Dellagostin3, João Carlos Deschamps1, 5
Tiago Collares1* 6
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1Laboratório de Embriologia Molecular e Transgênese, 2In Vitro Sul Ltd. 8
Pesquisa e Desenvolvimento, 3Laboratório de Biologia Molecular, Núcleo de 9
Biotecnologia, Centro de Desenvolvimento Tecnológico, Universidade Federal 10
de Pelotas, Pelotas, RS 11
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*Corresponding author: Centro de Desenvolvimento Tecnológico, Universidade 13
Federal de Pelotas, Campus Universitário s/nº, CEP 96010-900, Caixa Postal 14
354, Pelotas, RS, Brazil, Phone: +55 53 3275 7588, Fax: +55 3275 7551, E-15
mail: [email protected] 16
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Abstract 1
The objective was to introduce exogenous DNA into commercially sex-2
sorted bovine sperm using nanopolymer for transfection. In the first experiment, 3
the optimal concentration and ratio of linear-to-circular plasmid was determined 4
for NanoSMGT in unsorted sperm. A second experiment was conducted to 5
transfect exogenous DNA into sex-sorted sperm. Exogenous DNA uptake 6
occurred in a dose-dependent manner (P<0.05). The optimal amount of DNA 7
was 10 µg/106 cells. The ratios of linear-to-circular plasmid do not influence the 8
uptake by unsorted sperm cells and none of the tested treatments affected 9
sperm motility and viability. Commercially sex-sorted bovine sperm were able to 10
uptake exogenous DNA using nanopolymer; however, both X- and Y-sorted 11
sperm had decreased DNA uptake in comparison to unsorted sperm (P<0.05). 12
Neither sperm motility nor viability were affected by nanotransfection. In 13
conclusion, nanopolymer efficiently introduced exogenous DNA into 14
commercially sex-sorted bovine sperm; we inferred that these sperm could be 15
used for production of embryos of the desired sex, a technique named 16
NanoSMGT. 17
18
Keywords: NanoSMGT; Nanopolymer; Sperm-mediated gene transfer; Sexed 19
sperm; Cattle 20
21
22
1. Introduction 1
2
Sperm-mediated gene transfer (SMGT) has been applied to transgenesis 3
in many species [1-3]. This technique could become the most efficient and cost-4
effective technique to generate transgenic animals, which would substantially 5
increase their application in biomedical research and in commercial production, 6
although, its efficiency needs to be improved [4,5]. Several studies have 7
reported strategies to improve DNA uptake by sperm, including electroporation 8
[6], lipofection [7], and DMSO/DNA complex [8], although generation of 9
offspring was still low [9]. However, the successful use of nanostructures to 10
introduce foreign DNA into eukaryotic cells [10-12] has brought new 11
perspectives for production of transgenic embryos. Recently, Kim et al. [13] 12
obtained high transgenic rates for swine embryos after IVF using sperm 13
transfected with magnetic nanoparticles. 14
Bovine transgenesis has been exploited for several purposes, including 15
production of pharmaceutical proteins in cows [14], or increased muscle mass 16
in cattle [15]. The microinjection of plasmid DNA into early embryos represents 17
the state of the art in generating transgenic cattle. However, this approach 18
suffers from substantial drawbacks (e.g. mosaic distribution of the injected 19
transgene, late transgene integration at high copy numbers, and low 20
transgenesis frequency), making generation of transgenic lines a laborious task 21
[16]. Conversely, mass generation techniques, such as SMGT, have been 22
widely used to produce transgenic bovine embryos [5,7,17,18]. Thus, according 23
to the application of transgenic cattle, embryo sex selection becomes 24
necessary. Bovine sex-sorted sperm have been commercialized and 25
23
successfully used for production of embryos of the desired sex, without the 1
disadvantages of an embryo biopsy [19]. Today, high quality sex-sorted bovine 2
sperm is available in several countries at relatively low costs [20]. Recently, De 3
Cecco et al., [21] demonstrated the use of sex sorted sperm for production of 4
transgenic swine embryos. 5
The objective of the present study was to determine the ability of 6
commercially available cryopreserved sorted X and Y bovine sperm to uptake 7
exogenous DNA using nanopolymer as transfectant, a method named 8
NanoSMGT. 9
10
2. Materials and methods 11
12
2.1. Sperm source 13
14
Bovine sperm (X-sorted, Y-sorted and unsorted), from three Nelore bulls, 15
was purchased from CRV Lagoa Ltd. (São Paulo, SP, Brazil). According to the 16
supplier, there were 2 x 106 sperm in each sex-sorted dose, and 15 x 106 sperm 17
per straw of unsorted control semen. 18
19
2.2. Sperm nanotransfection 20
21
One frozen semen straw from each of the three bulls was thawed in a 22
water bath (35 °C for 30 s) and then pooled. Sperm were separated from 23
commercial cryopreservation media by centrifugation for 5 min at 250 × g, 24
suspended and maintained in Opti-MEM I medium (Invitrogen, Carlsbad, CA, 25
24
USA). Plasmid pEGFP-N1 (4.7 kb; Catalog #6085-1, Clontech Laboratories 1
Inc., Mountain View, CA, USA) was mixed with NanoFect Transfection Reagent 2
(Qiagen, Mississauga, ON, Canada) according to manufacturers’ instructions, 3
and incubated with sperm (1 × 106 cells) for 60 min at 38.5 °C and 5% (v/v) CO2 4
in air with high humidity. After incubation, sperm were washed three times in 5
OptiMEM I medium by centrifugation for 5 min at 600 × g and incubated with 20 6
U of DNase I (Invitrogen) for 30 min to remove exogenous DNA adsorbed on 7
the sperm surface, but not internalized. After DNase I treatment, sperm were 8
washed three more times in PBS (Ca2+ and Mg free) by centrifugation for 5 min 9
at 600 × g and the sperm pellet was subjected to DNA extraction. 10
11
2.3. Sperm motility and sperm viability analyses 12
13
Sperm motility was visually assessed in approximately 4-6 fields of 14
approximately 100 sperm each under a phase contrast microscope. Motility was 15
expressed as the average percentage of forward motile sperm of each sample. 16
Sperm viability was evaluated using the LIVE/DEAD® Sperm Viability Kit 17
(Invitrogen), according to manufacturer’s protocol. The number of red (dead) 18
and green cells (live) in a total of 100 sperm was counted in triplicate for each 19
sample under a fluorescence microscope. Viability was expressed as the 20
average percentage of viable sperm cells. Sperm motility and viability were 21
evaluated before and immediately after DNA incubation with sperm cells. All 22
evaluations were done independently by three persons, and data were 23
averaged. 24
25
25
2.4. Evaluation of DNA uptake by quantitative PCR 1
2
Template DNA for real-time PCR quantification was extracted from 3
nanotransfected sperm using the DNeasy Blood & Tissue Kit (Qiagen), 4
according to manufacturer’s protocol. Reactions were run using 1 ng of 5
template DNA on a Stratagene® Mx3005P™ Real-Time PCR System (Agilent 6
Technologies, Santa Clara, CA, USA), using Platinum® SYBR® Green qPCR 7
SuperMix UDG (Invitrogen). Primers for pEGFP vector (forward 5’ 8
CACGTCATTTTCCTCCTGCAT 3’, reverse 5’ GCATAGCGGCTCGTAGAGGTA 9
3’ were designed with Primer3 software (http://frodo.wi.mit.edu/primer3). Initial 10
validation experiments were conducted to ensure that primers had optimal PCR 11
efficiency. For quantification, a standard curve (Fig. 1) was generated using a 12
serial dilution of pEGFP plasmid (102 to 107 copies). Amplification was carried 13
out at cycling conditions of 95 ºC for 2 min, followed by 40 cycles at 95 ºC for 15 14
s, 51 ºC for 30 s, 72 ºC for 30 s followed by conditions to calculate the melting 15
curve. All PCR runs were performed in duplicate. 16
17
2.5. Experimental design 18
19
2.5.1. Experiment 1: Determination of optimal concentration and ratio of linear-20
to-circular plasmid for nanotransfection in unsorted sperm 21
22
In order to determine the most efficient nanotransfection protocol, sperm 23
were incubated with 0.1, 1, and 10 µg of exogenous DNA. Also, for each 24
plasmid concentration, three linear-to-circular DNA ratios were tested: only 25
26
linear (L), linear and circular (1:1; L:C), and only circular (C). The pEGFP vector 1
was linearized with the restriction enzyme NotI and purified with GFX PCR DNA 2
and Gel Band Purification Kit (GE Healthcare©, Buckinghamshire, UK). Sperm 3
motility and viability were evaluated before and after incubation, followed by 4
quantification of exogenous DNA uptake by sperm cells. This experiment was 5
replicated three times using separate pools of frozen-thawed semen. 6
7
2.5.2. Experiment 2: Exogenous DNA nanotransfection of sex-sorted sperm 8
9
The objective of this experiment was to investigate the ability of sex-10
sorted bovine sperm to uptake exogenous DNA after nanopolymer transfection. 11
Using the most efficient plasmid concentration in the previous experiment, X-12
sorted, Y-sorted, and unsorted sperm were incubated with three linear-to-13
circular DNA ratios, as described above. Sperm incubated without exogenous 14
DNA and nanopolymer for each group was used as a control. Sperm motility, 15
viability, and quantification of DNA uptake were performed as previously 16
described. This experiment was repeated three times using separate pools of 17
frozen-thawed semen. 18
19
2.6. Data analyses 20
21
Linear regression was used to evaluate the standard curve of plasmid 22
serial dilution. Sperm motility and viability, before and after incubation, were 23
compared using a Student’s paired t-test. That the data were normally 24
distributed was verified using a Kolmogorov-Smirnov’s test. In Experiment one, 25
27
a square root transformation was performed to normalize DNA uptake data. 1
Quantification of exogenous DNA uptake was compared using two-way 2
ANOVA, followed by a Tukey test for multiple comparisons. The parameters 3
evaluated in the first experiment were “linear-to-circular plasmid ratio (L, L:C 4
and C)” and “exogenous DNA concentration”. The parameters evaluated in the 5
second experiment were “sperm type (Unsorted, X-sorted and Y-sorted)” and 6
“linear-to-circular plasmid ratio (L, L:C and C)”. Significance was considered at 7
P<0.05 in all analyses. All data were expressed as mean ± SEM. 8
9
3. Results 10
11
3.1. Exogenous DNA optimal concentration and ratio of linear-to-circular 12
plasmid in unsorted sperm 13
14
The use of nanopolymer was efficient for sperm transfection. The linear-15
to-circular plasmid ratio did not affect the uptake of exogenous DNA (P = 16
0.2453). However, exogenous DNA uptake occurred in a dose-dependent 17
manner, increasing as availability of DNA for transfection increased (P<0.0001, 18
Fig. 2a). Only linearized plasmid DNA at a concentration of 1 µg resulted in 19
reduced sperm viability (P<0.05, Fig. 2c). No significant differences were found 20
in other treatments regarding sperm motility and viability evaluated before and 21
immediately after incubation with exogenous DNA, even at high DNA 22
concentrations, or various linear-to-circular plasmid ratios (Fig. 2b, 2c). 23
24
3.2. Uptake of exogenous DNA by sex-sorted sperm 25
28
1
From the results of previous experiment, 10 µg of plasmid was chosen to 2
test the optimal ratio of linear-to-circular plasmid to maximize the uptake of 3
exogenous DNA by commercially sex-sorted sperm, using nanopolymer. The 4
ratio of linear-to-circular plasmid did not affect the uptake of exogenous DNA, 5
independent of the sorting procedure (P = 0.9188). Both X- and Y-sorted sperm 6
had lower uptake of plasmids (P = 0.0004) in comparison to unsorted sperm. 7
However, in X-sorted sperm incubated with circular plasmid, uptake was similar 8
to unsorted sperm (Fig. 3a). In X-sorted sperm, sperm motility was reduced 9
after incubation with circular DNA; conversely, motility was also reduced for 10
sperm incubated without exogenous DNA (Control, Fig. 3b). Similarly, X-sorted 11
sperm, treated with any linear-to-circular plasmid ratio or not treated (control) 12
had reduced sperm viability (Fig. 3c). We inferred that this reduction was 13
caused by incubation time and not by exogenous DNA, since sperm motility and 14
viability was also reduced in controls. As demonstrated for unsorted sperm, in 15
other treatments sperm motility and sperm viability were not affected by 16
exogenous DNA (Fig. 3b, 3c). 17
18
4. Discussion 19
20
In the current study, the efficient introduction of exogenous DNA into sex-21
sorted bovine sperm using nanopolymer as transfectant was demonstrated. To 22
our knowledge, this is the first report of bovine SMGT using commercially sex-23
sorted sperm associated to nanocomposites for exogenous DNA transfection. In 24
the past decade, several cationic nanopolymers have been demonstrated to 25
29
display considerable transfection efficiency in vivo and in vitro [22]. Generally 1
they have abundant primary amines which readily form polyplexes with 2
negatively charged DNA and subsequently buffer the endosomal environment, 3
facilitating the release of DNA into the cytosol [23]. Previously, magnetic 4
nanoparticles were successfully used to promote introduction of exogenous 5
DNA into boar sperm and subsequently production of transgenic embryos [13]. 6
Several studies on bovine SMGT have reported divergences on 7
exogenous DNA concentration and its uptake by sperm, ranging from 0.1 to 10 8
µg [5,7,18]. Conversely, other approaches have been tested to increase DNA 9
uptake, such as the use of liposomes [7]. In the present study, using 10
nanopolymer, 10 µg/106 cells was the optimal concentration of DNA for 11
unsorted sperm nanotransfection. In previous reports, large amounts of 12
exogenous DNA associated with sperm triggered endonuclease activation, 13
resulting in exogenous and endogenous DNA degradation in an apoptosis-like 14
process [18,19,24,25]. However, exogenous DNA did not induce DNA 15
fragmentation in bovine sperm [18]. Furthermore, Anzar and Buhr [5] reported 16
decreases bovine sperm viability using 0.5 µg/0.25 x 106 cells. However, in the 17
present study, even the highest DNA concentration used did not decrease 18
sperm motility and viability; therefore, we inferred that nanotransfected sperm 19
could be used for IVF or even AI. 20
As suggested by Li et al. [20,26] and Collares et al., [2] the ratio of linear-21
to-circular DNA can influence exogenous DNA uptake by sperm cells. In the 22
present study, nanopolymer complexed with linear, a mixture of linear and 23
circular, or circular plasmid did not influence the amount of DNA uptake. These 24
30
results were noteworthy, since synthetic constructions that sometimes are only 1
available as linear structure could be used without affecting DNA internalization. 2
Interestingly, the uptake of exogenous DNA by sex sorted sperm was 3
reduced in comparison to unsorted sperm. Sexing sperm by high-speed flow-4
cytometry subjects them to various stresses, including high dilution, Hoechst 5
nuclear staining, high pressure, mechanical forces associated with passage 6
through the sorter, exposure to UV laser beam, electrical charge, and projection 7
into the collection tube at high speed [27-30]. As demonstrated by Spinaci et al. 8
[31], sperm sorting induces a re-distribution of hsp70, a protein that plays a role 9
during sperm-oocyte membrane interaction [32]. In mammals, 30-35 kDa sperm 10
proteins positively interact with exogenous DNA, allowing internalization by the 11
sperm nucleus [33]. Perhaps sperm proteins other than 30-35 kDa may be 12
affected by the sorting procedure, potentially changing the localization of these 13
proteins, and interfering with DNA internalization by sperm. Future studies 14
should be conducted to elucidate these mechanisms. 15
De Cecco et al. [21] did not evaluate the incorporation of exogenous 16
DNA for Y- or X-sorted; however, they reported that sorted sperm treated with 17
exogenous DNA was able to produce transgenic embryos. Therefore, we 18
inferred that the lower DNA uptake in sex-sorted sperm should not interfere with 19
production of sex-selected transgenic bovine embryos by NanoSMGT, since 20
previous studies reported production of transgenic embryos with sperm that had 21
taken up small amounts of exogenous DNA [3]. In addition, in recent studies, 22
nanotransfectants protected DNA strands against nucleases during cellular 23
delivery [34]. Specifically, DNA vector were protected from enzymatic cleavage 24
and interference from nucleic acid binding proteins, increasing the transgene 25
31
internalization success. Our nanotransfection protocol did not decrease sperm 1
motility and viability of sorted sperm. The reduction of sperm viability in the X-2
sorted sperm after incubation was not attributed to exogenous DNA, since the 3
control group also had a decreased number of viable sperm; perhaps this 4
reduction was caused by incubation time, as previously demonstrated [35]. 5
In summary, our study demonstrated for the first time the use of 6
nanopolymer to introduce exogenous DNA into bovine sex-sorted sperm. There 7
was no loss of sperm motility and viability; therefore, we concluded that 8
NanoSMGT could be used for production of sex-selected, transgenic bovine 9
embryos. 10
11
Acknowledgements 12
13
This work was supported by Brazilian CNPq and FAPERGS. Both V. F. 14
Campos and P.M. de Leon are students of the Graduate Program in 15
Biotechnology at Universidade Federal de Pelotas and are supported by 16
Brazilian CAPES, whereas J.C. Deschamps and O.A. Dellagostin are research 17
fellows of CNPq. 18
19
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Figure captions 1
2
Figure 1. Standard curve of serial dilution of pEGFP plasmid (102 to 10
7 copies). The slope, 3
regression coefficient, and primer efficiency are shown. 4
5
102 103 104 105 106 107
0
5
10
15
20
25
30
35
40
Slope = - 3.25, r2 = 0.999, Eff = 103%
Initial quantity (copies)
Cyc
le t
hre
sh
old
(C
t)
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
37
Fig. 2. Nanotransfection in unsorted sperm. In all figures, data are expressed as means ± SEM 1
(N = 3). a) Evaluation of DNA uptake by quantitative PCR at various concentrations and ratios 2
of linear-to-circular plasmid, showing increases in DNA uptake in a dose-dependent manner. 3
Amplification in the control group without exogenous DNA was not detected. a-c
Differences 4
among amounts of exogenous DNA (P<0.0001). There were no differences (P=0.2453) were 5
among various linear-to-circular ratios in all treatments. This graph has square root transformed 6
data. b) Sperm motility before and after incubation with exogenous DNA. c) Sperm viability 7
before and after incubation with exogenous DNA. *Difference (P<0.05) between means before 8
and after incubation. 9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
Ctrl 0.1 1 100
510 01
110 02
210 02 Linear
Linear : Circular
Circular
a
b
c
DNA quantity (g)
Inte
rnaliz
ed p
lasm
ids/ 10
6 s
perm
Ctrl L L:C C L L:C C L L:C C0
20
40
60
80Before DNA incubation
After DNA incubation
0.1 g 1 g 10 g
Treatments
Sp
erm
mo
tilit
y (
%)
Ctrl L L:C C L L:C C L L:C C0
20
40
60
80Before DNA incubation
After DNA incubation
0.1 g 1 g 10 g
*
Treatments
Sp
erm
via
bili
ty (
%)
a)
b)
c)
38
Fig. 3. Nanotransfection in sex-sorted spermatozoa. Data are expressed as means ± SEM (N = 1
3). a) Evaluation of DNA uptake by quantitative PCR at various ratios of linear-to-circular 2
plasmid in unsorted, X and Y sorted sperm, with reduction in DNA uptake by sex-sorted sperm. 3
Amplification in the control group (without exogenous DNA) was not detected, thus they do not 4
appear in the graph. A,B
Treatments without a common superscript differ (P=0.0004). a,b
Within 5
linear-to-circular plasmid ratios, numbers without a common superscript differ (P=0.0004). b) 6
Sperm motility before and after incubation with exogenous DNA. c) Sperm viability before and 7
after incubation with exogenous DNA. *Difference (P<0.05) between means before and after 8
incubation. 9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
Unsorted X - sorted Y - sorted0
110 08
210 08
310 08
Linear
Linear : Circular
CircularAa
Aa
Aa
Bb
Bb
Bb
Bb Bb
Bb
Treatments
Inte
rnaliz
ed p
lasm
ids/ 10
6 s
perm
Ctrl L L:C C Ctrl L L:C C Ctrl L L:C C0
20
40
60
80 Before DNA incubation
After DNA incubation
Unsorted X - sorted
**
Y - sorted
Treatments
Sp
erm
mo
tilit
y (
%)
Ctrl L L:C C Ctrl L L:C C Ctrl L L:C C0
20
40
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Before DNA incubation
After DNA incubation
Unsorted X - sorted Y -sorted
* ** *
Treatments
Sp
erm
via
bili
ty (
%)
a)
b)
c)
39
1
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3
4
5
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7
3. ARTIGO 2 8
9
10
11
12
NanoSMGT: transgene transmission into bovine embryos using halloysite 13
clay nanotubes or nanopolymer to improve transfection efficiency 14
15
(Publicado no periódico Theriogenology, in press) 16
17
18
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24
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40
NanoSMGT: transgene transmission into bovine embryos using halloysite 1
clay nanotubes or nanopolymer to improve transfection efficiency 2
3
Vinicius Farias Campos1, Priscila Marques Moura de Leon1, Eliza Rossi 4
Komninou1, Fabiana Kömmling Seixas2, Odir Antônio Dellagostin3, João Carlos 5
Deschamps1, Tiago Collares1* 6
7
1Laboratório de Embriologia Molecular e Transgênese, Núcleo de 8
Biotecnologia, Centro de Desenvolvimento Tecnológico, Universidade Federal 9
de Pelotas, Pelotas, RS, Brazil 10
2Laboratório de Genômica Funcional, Núcleo de Biotecnologia, Centro de 11
Desenvolvimento Tecnológico, Universidade Federal de Pelotas, Pelotas, RS, 12
Brazil 13
3Laboratório de Biologia Molecular, Núcleo de Biotecnologia, Centro de 14
Desenvolvimento Tecnológico, Universidade Federal de Pelotas, Pelotas, RS, 15
Brazil 16
17
*Corresponding author: Centro de Desenvolvimento Tecnológico, Universidade 18
Federal de Pelotas, Campus Universitário s/nº, CEP 96010-900, Caixa Postal 19
354, Pelotas, RS, Brazil, Phone: +55 53 3275 7588, Fax: +55 3275 7551, E-20
mail: [email protected] 21
41
Abstract 1
The objectives were to investigate whether: 1) nanotransfectants are more 2
effective than other common transfection methods for SMGT; 2) NanoSMGT is able 3
to transmit exogenous DNA molecules to bovine embryos; and 3) halloysite clay 4
nanotubes (HCNs) can be used as a transfection reagent to improve transgene 5
transmission. Four transfection systems were used: naked DNA (without 6
transfectant), lipofection, nanopolymer, and halloysite clay nanotubes. Plasmid 7
uptake by sperm and its transfer to embryos were quantified by conventional and 8
real-time PCR, as well as EGFP expression by florescence microscopy. Furthermore, 9
sperm motility and viability, and embryo development were investigated. Mean 10
number of plasmids taken up was affected (P<0.05) by transfection procedure, with 11
the nanopolymer being the most effective transfectant (~ 153 plasmids per 12
spermatozoon). None of the treatments affected sperm motility or viability. The mean 13
number of plasmids transmitted to four-cell stage embryos was higher (P<0.05) in 14
nanopolymer and HCNs than liposomes and naked DNA groups. The number of 15
embryos carrying the transgene increased from 8-10% using naked DNA or 16
liposomes to 40-45% using nanopolymer or HCN as transfectants (P<0.05). There 17
were no significant differences among transfection procedures regarding blastocyst 18
formation rate of resulting embryos. However, no EGFP-expressing embryo was 19
identified in any treatment. Therefore, nanotransfectants improved transgene 20
transmission in bovine embryos without deleterious effects on embryo development. 21
To our knowledge, this was the first time that bovine embryos carrying a transgene 22
were produced by NanoSMGT. 23
24
Keywords: SMGT; Halloysite clay nanotubes; Nanopolymer; Cattle; Exogenous DNA 25
26
42
1. Introduction 1
2
Over the past 30 y, several methods to generate transgenic animals have 3
been developed [1-3]. The most common methods have been pronuclear 4
microinjection, somatic cell nuclear transfer, retroviral vectors, and most recently, 5
embryonic-stem cell transgenesis. The use of sperm for transgenesis has been 6
studied, and several distinct approaches have been developed; however, the 7
efficiency of such sperm-mediated gene transfer (SMGT) needs to be radically 8
improved [4,5]. The limited uptake of exogenous DNA and its subsequent 9
degradation by sperm, remain the primary factors underlying the low efficiency of this 10
technique [6-9]. 11
Several approaches have been utilized in order to improve DNA uptake. 12
Methods such as electroporation, lipofection, DNA/DMSO complexes and restriction-13
enzyme-mediated integration (REMI) have been used; however the frequency of 14
transgenic offspring remains low [10-13]. Conversely, the more recent use of 15
nanotechnology in the context of SMGT has new possibilities for the efficient delivery 16
of exogenous DNA into sperm. For example, Kim et al [14] obtained high transgenic 17
rates for swine embryos after in vitro fertilization using sperm transfected with 18
magnetic nanoparticles. Recently, we successfully demonstrated exogenous DNA 19
uptake by bovine sperm using a nanopolymer as the transfectant, providing a new 20
tool to improve transgenic bovine embryo production [11]. 21
Functional nanometer-scale containers have been successfully used to deliver 22
pharmaceuticals and nucleic acid molecules into animal cells and tissues. In this 23
regard, halloysite clay nanotubes (HCNs) are of particular interest. They are a natural 24
nanomaterial, with deposits in several countries, including the USA and Brazil [15]. 25
43
Halloysite clay nanotubes (HCNs) are biocompatible and spontaneously incorporated 1
by cells. The diameter of their inner lumen is compatible with many macromolecules 2
and proteins, allowing the entrapping and subsequent slow release of a range of 3
active agents [16]. These characteristics make this nanomaterial a potentially 4
functional transfection agent for use in bovine SMGT. 5
Bovine transgenesis has been exploited for several purposes, including 6
production of pharmaceutical proteins in cows [17] and increased muscle mass in 7
cattle [18]. The microinjection of plasmid DNA into early embryos represents the state 8
of the art in generating transgenic cattle. However, this approach has substantial 9
drawbacks (e.g. mosaic distribution of the injected transgene, late transgene 10
integration at high copy numbers, and low transgenesis frequency), making 11
generation of transgenic lines laborious [19]. Conversely, mass generation 12
techniques, such as SMGT, represent a potentially important alternative means of 13
producing transgenic bovine embryos [6,11,20-23]. 14
Based on our previous results demonstrating that NanoSMGT can be an 15
effective technique for the introduction of exogenous DNA into bovine sperm [11], our 16
objective in the present study was to transmit a transgene to bovine embryos through 17
NanoSMGT, using nanopolymer or HCNs as transfection agents. 18
19
2. Materials and methods 20
21
2.1. Sperm source and experimental design 22
23
Sperm from three Nelore bulls was purchased from CRV Lagoa Ltd. (São 24
Paulo, SP, Brazil). According to the supplier, there were 15 x 106 sperm per straw. 25
44
1
2.2. Sperm transfection procedures 2
3
One frozen semen straw from each of the three bulls was thawed in a water 4
bath (35 °C for 30 s) and all semen pooled. Sperm were separated from commercial 5
cryopreservation media by centrifugation for 5 min at 250 × g, suspended and 6
maintained in Opti-MEM I medium (Invitrogen, Carlsbad, CA, USA). For all 7
transfection procedures, plasmid pEGFP-N1 (4.7 kb; Catalog #6085-1, Clontech 8
Laboratories Inc., Mountain View, CA, USA) was used at 10 µg of final quantity and 9
1:1 (linear:circular) plasmid ratio, the best arrangement for exogenous DNA uptake 10
by bovine sperm [11]. Linearization was performed using the restriction enzyme NotI 11
and plasmid was purified with GFX PCR DNA and Gel Band Purification Kit (GE 12
Healthcare©, Buckinghamshire, UK). Sperm transfection was performed using the 13
same sperm pool at 1 × 106 sperm per transfection procedure. Four transfection 14
treatments were conducted: 1) naked exogenous DNA (without any trasfectant), 2) 15
lipofection using Lipofectamine 2000 (Invitrogen), 3) transfection using nanopolymer 16
(NanoFect Transfection Reagent, Qiagen, Mississauga, ON, Canada), and 4) 17
halloysite clay nanotubes (HCNs; Sigma-Aldrich, St. Louis, MO, USA). Also, sperm 18
without DNA transfection was used for IVF as a control group. 19
20
2.2.1. Transfection using Liposome 21
22
Lipofectamine 2000 was prepared according the manufacturer's instructions. 23
Two solutions were prepared: Solution A: 10 μg of 1:1 linear-to-circular plasmid 24
ratio/50 μL Opti-MEM I; and Solution B: 10 μg Lipofectamine/50 μL Opti-MEM I. 25
45
Solutions A and B (50 μL each) were combined in 500 µL tubes for 1 h at ambient 1
temperature to allow DNA-liposome complexes to form. After incubation, the resultant 2
complexes were combined with a suspension of sperm (106 cells/100 μL OptiMEM I) 3
for a total volume of 200 μL, followed by gentle mixing and a 1 h incubation at 38.5 4
ºC with 5% (v/v) CO2 in air with high humidity. To separate sperm from DNA which 5
remained unattached to the sperm membrane, sperm were centrifuged (5 min at 600 6
× g) and washed three times using Opti-MEM I medium. In addition, sperm were 7
incubated with 20 U of DNase I (Invitrogen) for 30 min and then re-suspended into 15 8
µL of Opti-MEM I medium prior to IVF. Alternatively, sperm were washed in PBS 9
(Ca2+ and Mg free) by centrifugation for 5 min at 600 × g, with the sperm pellet 10
subjected to DNA extraction for plasmid uptake quantification. 11
12
2.2.2. Transfection using NanoFect 13
14
Sperm and exogenous DNA were prepared as described above. NanoFect 15
transfection was performed as previously described [11]. Sperm were separated from 16
transfection media as described above. 17
18
2.2.3. Transfection using halloysite clay nanotubes (HCNs) 19
20
Sperm and exogenous DNA were prepared as described above. The HCNs 21
were diluted in OptiMEM I medium at 75 µg/mL, as described [16]. As described 22
above for Lipofectamine transfection, two solutions were prepared: Solution A: 10 μg 23
of 1:1 linear-to-circular plasmid ratio/50 μL Opti-MEM I; and Solution B: 50 µL of 75 24
µg/mL HCNs diluted in Opti-MEM I. Solutions A and B (50 μL each) were combined 25
46
in 500 µL tubes for 1 h at ambient temperature. After incubation, the resultant 1
complex was combined and separated from sperm as described above. 2
3
2.2.4. Transfection without transfectant 4
5
Sperm and exogenous DNA were prepared as described above. A solution 6
containg 10 μg of 1:1 linear-to-circular plasmid ratio diluted in 100 µL of OptiMEM I 7
medium was combined and separated from sperm, as described above. 8
9
2.3. Sperm motility and sperm viability analyses 10
11
Sperm motility and sperm viability were determined as described [11]. Briefly, 12
sperm motility was assessed in four to six fields of approximately 100 sperm each 13
under a phase contrast microscope. Sperm viability was evaluated using the 14
LIVE/DEAD® Sperm Viability Kit (Invitrogen), according to the manufacturer’s 15
protocol. Sperm motility and viability were evaluated before and immediately after 16
DNA incubation with sperm. All evaluations were done independently by three 17
persons, and data were averaged. 18
19
2.3. Quantification of the number of internalized plasmids by sperm cells 20
21
Quantification of DNA uptake by sperm cells after transfection procedures was 22
conducted as reported [11]. Briefly, template DNA for real-time PCR quantification 23
was extracted from nanotransfected sperm using the DNeasy Blood & Tissue Kit 24
(Qiagen), according to the manufacturer’s protocol. Reactions were run on a 25
47
Stratagene® Mx3005P™ Real-Time PCR System (Agilent Technologies, Santa Clara, 1
CA, USA), using Platinum® SYBR® Green qPCR SuperMix UDG (Invitrogen) using 2
primers for pEGFP vector (forward 5’ CACGTCATTTTCCTCCTGCAT 3’, reverse 5’ 3
GCATAGCGGCTCGTAGAGGTA 3’). For quantification, a standard curve was 4
generated using a serial dilution of pEGFP plasmid (101 to 107 copies). Amplification 5
was carried out at cycling conditions of 95 ºC for 2 min, followed by 40 cycles at 95 6
ºC for 15 s, 51 ºC for 30 s, 72 ºC for 30 s, followed by conditions to calculate the 7
melting curve. All PCR runs were performed in duplicate. 8
9
2.4. In vitro fertilization trials 10
11
2.4.1. Oocyte collection and in vitro maturation (IVM) 12
13
The ovaries were obtained from a local abbatoir. Cumulus-oocyte complexes 14
(COCs) were aspirated from follicles (2-8 mm in diameter) with a sterile 18-guage 15
needle attached to a disposable syringe. All oocytes with a homogeneous cytoplasm 16
and with at least three intact layers of surrounding cumulus cells were selected for 17
IVM. Prior to IVM, COCs were washed three times in TCM-199 HEPES (Gibco Life 18
Technologies, Grand Island, NY, USA) supplemented with 10% FCS and 50 g 19
gentamycin sulfate, and were washed once in bicarbonate TCM-199 (Gibco Life 20
Technologies) supplemented with 10% FCS, 5 µg LH (Ayerst, Rouses Point, NY, 21
USA), 0.5 µg FSH (Folltropin, Vetrepharm, Belleville, ON, Canada), 1 µg estradiol 22
(estradiol-17β, Sigma E-8875), 2.2 µg pyruvate (Sigma P-4562), and 50 µg 23
gentamycin/mL of medium. Group of COCs (maximum 20) were matured in 90 µL of 24
48
the same medium droplet under mineral oil at 38.5 ºC, 5% CO2 in air with maximum 1
humidity for 22-24 h. 2
3
2.4.2. In vitro fertilization 4
5
COCs were washed three times in TALP fertilization with 25 mM HEPES 6
(Gibco Life Technologies, Grand Island, NY, USA) and 0.3% BSA (Sigma A-9647), 7
and were washed once in TALP fertilization medium supplemented with 10 µg/mL 8
heparin and 160 µL PHE solution [24], without hormones and other components of 9
maturation medium. Then, oocytes were finally transferred in groups of up to 25 for 10
each IVF droplet, previously equilibrated in an incubator with a temperature of 38.5 11
ºC and an atmosphere of 5% CO2 in air for at least 1 h. Fertilization was conducted 12
using treated sperm described above. Also, sperm from the same pool used for 13
transfection procedures was used for IVF without DNA transfection to serve as a 14
control group. Final concentration of sperm in fertilization droplets was adjusted to 1 15
× 106 cells/droplet. 16
17
2.4.3. In vitro culture 18
19
Eighteen hours after IVF, presumptive zygotes were stripped of cumulus cells 20
by repeated pipetting. Zygotes were washed three times in SOF culture medium 21
(SOFaa BSA, containing 8 mg/mL BSA [free of fatty acid] and 1 mM glutamine) for 22
removal of granulosa cells (denuded) and dead sperm. Zygotes surrounded by some 23
layers of granulosa cells were then transferred to plates in micro droplets with 100 µL 24
and cultured for 7 d in SOF culture medium at a temperature of 38.5 ºC in an 25
49
atmosphere of 5% CO2 in air with maximum humidity. The cleavage rate was 1
evaluated after 72 h of fertilization (Day 3), with removal of 50 μL of medium and 2
replacement with 50 µL of fresh medium. After 96 h of fertilization (Day 5), 50 μL of 3
medium was again replaced, with the addition of 1 µg/mL glucose. The rate of 4
blastocyst production was evaluated at Day 7. The osmolarity was maintained at 270 5
to 280 mOsmol and the pH was 7.4. 6
7
2.4.4. Embryo evaluation 8
9
During culture, embryos were assessed daily for morphological stage of 10
development, 1 to 7 d after being transferred to culture media. The assessment 11
criteria were successive stages of embryo development: 2-8 blastomeres (cleaved), 12
8-16 blastomeres, morula, and blastocyst. In addition, embryos were evaluated for 13
the presence of pEGFP-N1 plasmid by PCR analysis, quantification of transmitted 14
plasmids by quantitative PCR, and transgene expression by fluorescence 15
microscopy. 16
17
2.5. Transgene evaluation 18
19
2.5.1. Transngene detection by PCR analysis of embryos 20
21
Cleaved to blastocyst embryos were used for PCR analysis. Embryos were 22
washed in PBS and transferred into 5 mg/mL pronase (Sigma-Aldrich, Sao Paulo, 23
SP, Brazil) in PBS for 1 min to remove the zona pellucida and any attached sperm. 24
This was considered necessary to avoid any potential contamination in the PCR 25
50
positive results, as a result of accessory sperm attached to the zona, since sperm 1
were transfected with exogenous DNA. Embryos were then washed three or four 2
times in PBS and stored in liquid nitrogen. To increase PCR efficiency, embryos were 3
digested with 2 μL of 10 mg/mL proteinase K (Invitrogen) at 65 ºC for 2 h. After 4
digestion, proteinase K was inactivated at 95 ºC for 10 min. To ensure embryo 5
digestion was successful, half of the digestion reaction was used for first PCR that 6
was conducted with control primers of bovine 1.715 satellite (5’-7
TGGAAGCAAAGAACCCCGCT-3’and 5’-TCGTCAGAAACCGCACACTG-3’), located 8
on an autosome, that produces a PCR product of 216 bp, indicating the success of 9
the PCR procedure [25]. The other half of the digestion reaction employed a second 10
PCR to detect if pEGFP-N1 vector was transmitted to embryos. Primers (5'-11
CGGGACTTTCCAAAATGTCG -3' and 5'- GAAGATGGTGCGCTCCTGGA -3') were 12
designed to amplify a 500 bp fragment from the pEGFP-N1 plasmid. Both first and 13
second PCR conditions consisted of an initial denaturation step (2 min at 94 °C) 14
followed by 30 cycles of 1 min at 94 °C, 1 min at 51 °C, and 1 min at 72 °C. The last 15
cycle was followed by a final incubation of 7 min at 72 ºC [5]. A positive control was 16
used for both PCRs; it consisted of 1 pg of pEGFP-N1 plasmid and 1 ng of bovine 17
genomic DNA purified from blood cells for 1.715 satellite amplification. Also a NTC 18
(no template control) was included for each reaction. The PCR products were 19
analyzed on a 1.5 % agarose gel stained with ethidium bromide. Samples with a 20
1.715 satellite and pEGFP-specific PCR products were classified as PCR positive for 21
the transgene. 22
23
2.5.2. Quantification of transmitted plasmids to 4-cell stage embryos 24
25
51
As previously described [20], 4-cell embryos were used to quantify the number 1
of plasmids transmitted to embryos. Embryos were digested as described above and 2
the digestion product was employed as a template to qPCR which was conducted as 3
described above (Section 2.3). 4
5
2.5.3. Evaluation of transgene EGFP expression in embryos 6
7
Embryos were examined by epifluorescence microscopy with fluorescein 8
isothiocyanate filters (excitation range of 395-470 nm and emission spectrum of 509 9
nm). This enabled the clear identification of non-fluorescent (non-EGFP-expressing) 10
and fluorescent embryos (EGFP expressing), which were scored accordingly. 11
12
2.6. Experimental design 13
14
2.6.1. Experiment 1: Effects of transfectants on exogenous DNA uptake by sperm 15
cells and sperm motility and viability 16
17
To compare nanotransfectants with most common transfection procedures, 18
sperm were transfected by four methods (naked exogenous DNA, Lipofectamine 19
2000, Nanofect, halloysite clay nanotubes) as described above. Also, non-20
transfected sperm were used as a control group. Sperm motility and viability were 21
evaluated before and after incubation, followed by quantification of exogenous DNA 22
uptake by sperm cells. This experiment was replicated three times using separate 23
pools of frozen-thawed semen. 24
25
52
2.6.2. Experiment 2: Effects of transfectants on transgene transmission and 1
expression in IVF embryos 2
3
Sperm were transfected by methods described above. Also, non-transfected 4
sperm were used for in vitro fertilization as a control. Treated and non-treated sperm 5
were subsequently used to fertilize in vitro-derived bovine oocytes at MII stage. 6
Cleavage and blastocyst formation rates of embryos were evaluated to determine if 7
transfectants affected in vitro embryo development. In addition, PCR analysis was 8
conducted to detect if the transgene was transmitted to embryos. Four-cell stage 9
embryos were used to quantify the number of transmitted plasmids to oocytes by 10
real-time PCR. Expression of EGFP was evaluated in all embryos by fluorescence 11
microscopy. All experimental procedures were repeated at least three times. 12
13
2.7. Data analyses 14
15
Sperm motility and viability, before and after incubation, were compared using 16
a Student’s paired t-test. That data were normally distributed was verified using a 17
Kolmogorov-Smirnov’s test. Exogenous DNA uptake by sperm cells after transfection 18
procedures and the number of transmitted plasmids to embryos was compared using 19
one-way ANOVA, followed by a Tukey’s test for multiple comparisons. Chi-square 20
analysis was performed to compare rates of cleavage, blastocyst formation and PCR 21
positivity among treatments. Significance was considered at P<0.05 in all analyses. 22
23
3. Results 24
25
53
3.1. Effects of transfectants on sperm motility, sperm viability and exogenous DNA 1
uptake by sperm cells 2
3
Sperm motility and viability were not affected by transfection procedure, since 4
no differences were found before and after the various transfection procedures 5
(P>0.05, Fig. 1a, b). Conversely, the number of plasmids internalized by sperm cells 6
was increased (P<0.05) when HCNs were used in relation to liposome or naked 7
DNA. Nanofect was the most effective transfectant (P<0.05), with a mean of 153 8
plasmids transfected by each spermatozoan (Fig. 3a). 9
10
3.2. Effects of nanotransfectants on embryo development 11
12
In all treatments, embryos reached the blastocyst stage at the expected time. 13
Zonae pellucidae were intact and their thickness was appropriate for expanded 14
embryos, inner cell masses were well identifiable at one pole, and blastomeres and 15
trophoblast cells appeared normal. In addition, rate of blastocyst formation was 16
similar among treatments (P = 0.462, Table 1). Moreover, there was an increase in 17
cleavage ratio (P = 0.012, Table 1) for embryos produced by sperm transfected with 18
halloysite clay nanotubes (HCN). 19
20
3.3. Effects of nanotransfectants on transgene transmission and expression 21
22
All PCR reactions were successfully conducted, since all PCR results for the 23
bovine 1.715 satellite produced a fragment of 216 bp. All transfection approaches 24
were able to transmit transgene sequences to embryos, based on detection of the 25
54
500 bp pEGFP-N1 transgene-specific PCR product fragment, whereas the control 1
group that was fertilized with non-transfected sperm did not have this fragment (Fig. 2
2). Rates of PCR positivity were 8.1, 10.2, 45.6, and 40.7% for naked DNA, liposome, 3
Nanofect, and HCNs respectively. The proportion of embryos carrying the transgene 4
(P<0.05) was higher in those fertilized with sperm transfected with Nanofect or HCNs 5
in comparison to those fertilized with sperm transfected with naked exogenous DNA 6
or liposomes (Table 1). Concomitant with these findings, there was an increase 7
(P<0.05) in the number of plasmids transmitted to oocytes when they were fertilized 8
with sperm transfected with Nanofect or HCNs, in comparison to groups fertilized with 9
sperm transfected with naked exogenous DNA or liposomes (Fig. 3b). No plasmid 10
copies were detected in the control embryos derived from non-transfected sperm 11
(control). None of the embryos expressed EGFP. 12
13
4. Discussion 14
15
In our previous study, nanopolymer efficiently transfected exogenous DNA into 16
bovine sperm [11]. The present study corroborated this result, since nanocomposites 17
improved transgene transmission to bovine embryos without adverse effects on 18
sperm motility or viability, or on in vitro embryo development. 19
Exogenous DNA uptake by sperm was significantly affected by transfection 20
procedure in the present study. There was significant improvement in transfection 21
efficiency for sperm using HCNs or nanopolymer in comparison to lipofection or 22
incubation with naked DNA. Whereas liposomes increased plasmid uptake [20,26], 23
transfection of bovine sperm with HCNs has apparently not been previously reported, 24
and consequently no comparison among liposomes, HCNs and nanopolymer has 25
55
been published. Kim et al. [14] demonstrated the successful production of transgenic 1
swine embryos using magnetic nanoparticles to introduce exogenous DNA into 2
sperm; however, their study did not include comparisons with other transfection 3
methods. 4
Lipofection transports plasmids across the plasma membrane by endocytosis. 5
However, before the internalized DNA molecules can reach the nucleus, their 6
intracellular trafficking has to proceed via the endosomal or lysosomal pathway, in 7
which a large number of the transgenes are hydrolyzed by nucleases. Similarly, when 8
naked exogenous DNA is used in SMGT, internalized transgenes are rapidly 9
degraded by DNases present in sperm [5,8,13,27]. Thus, by controlling the fate of 10
internalized plasmids, the intracellular trafficking process affected transformation 11
efficiency [28]. Additionally, DNases present in the seminal fluid can degrade 12
exogenous DNA molecules. Kim et al. [14] demonstrated that, when liposomes were 13
used, DNase I greatly reduced the rate of exogenous DNA binding to sperm, 14
whereas when magnetic nanoparticles were used, the reduction in binding was 15
significantly lower. Magnetic nanoparticles bound to exogenous DNA localized either 16
within the plasma membrane or on the plasma membrane, whereas most liposome-17
bound exogenous DNA localized on the plasma membrane [29]. Several cationic 18
nanopolymers displayed considerable transfection efficiency in vivo and in vitro [30]. 19
Generally, these agents have abundant primary amines, which readily form 20
polyplexes with negatively charged DNA and subsequently buffer the endosomal 21
environment, facilitating the release of DNA into the cytosol [31]. In addition, previous 22
studies have shown that nanotubes have the ability to protect bound DNA cargoes 23
(and other cargoes including DNA binding proteins) from enzymatic cleavage, both 24
during and after delivery into cells. Although the mechanisms of protection are not yet 25
56
well understood [32], it has been hypothesized that silica nanotubes can act as a 1
physical shield that protects the loaded materials from damage, since cellular 2
nucleases/proteins cannot physically access the DNA [33]. Studies using MCF-7 and 3
HeLa cells demonstrated that HCNs are spontaneously captured by cells and 4
become concentrated in the nuclear region [16]; therefore, nanotubes can be 5
internalized by cells and can deliver exogenous DNA directly to the nucleus. 6
Transgene transmission to 4-cell stage embryos was significantly affected by 7
the transfection procedure. When oocytes were fertilized with sperm treated with 8
HCNs or nanopolymer, the number of plasmids present in the embryos was 9
significantly increased, in comparison to sperm treated with naked DNA or liposomes. 10
Perhaps DNA uptake by sperm cells was significantly higher when HCNs or 11
nanopolymer are used. Indeed, the proportion of embryos carrying the transgene was 12
was significantly improved from 8-10% using naked DNA or liposomes to 40-45% 13
using HCNs or nanopolymer as transfection reagents. These findings were 14
compatible with the abovementioned hypothesis of protection, and helped to explain 15
the fact that, as demonstrated in the present study, nanocomposites such as HCNs 16
or Nanofect were more effective than naked DNA or liposomes in transmitting 17
transgene molecules to bovine embryos. 18
No EGFP-expressing embryos were obtained after IVF in any treatment. 19
Several researchers have investigated the use of bovine IVF-SMGT for transgenic 20
embryo production, but have reported low success in producing protein-expressing 21
embryos [20,34-36]. Here, we used a pEGFP-N1 vector that contains the CMV 22
promoter. Rieth et al. [36] also speculated about the low efficiency of the CMV 23
promoter; that an additional CMV enhancer improved EGFP expression and similar 24
effects are well known from experiments with injections into pronuclei [37,38] may be 25
57
also be an explanation. In addition, expression of EGFP-expression was not obtained 1
by IVF-SMGT in pigs [39] or sheep [40]. However, since production of embryos by 2
ICSI-SMGT resulted in EGFP expression, this aproach could be an alternative to IVF. 3
We believe that by using high transfection efficiency of nanocomposites, as 4
demonstrated herein, with improvement of ICSI-SMGT, the production of transgenic 5
bovine animals will be further enhanced, being an important next step for 6
NanoSMGT. 7
We previously demonstrated that nanopolymer had no effect on bovine sperm 8
motility and viability [11], thus establishing that it was biocompatible with these cells. 9
The present study further confirmed the biocompatibility of nanopolymer and HCNs in 10
the context of SMGT, since there were no deleterious effects on sperm motility and 11
viability, as well as on embryo survival or development using these transfection 12
agents. Similarly, transgenic bovine embryos produced by SMGT using liposomes as 13
transfection agent do display any altered embryo development [20,21]. Additionally, 14
in the present experiment, sperm motility during in vitro fertilization trials was not 15
significantly different among treatments. Furthermore, HCNs were biocompatible with 16
MCF-7 and HeLa cells, with cellular viability maintained up to 75 µg/ml HCNs [16]. In 17
the present study, this concentration was used without any negative effects on 18
embryo development. 19
In summary, our study demonstrated, apparently for the first time, the use of 20
nanopolymer or HCNs to improve transgene transmission to bovine embryos without 21
deleterious effects on embryo development. 22
23
Acknowledgements 24
25
58
The authors thank Dr. Kevin Smith for a critical review of the manuscript. This 1
work was supported by Brazilian CNPq and FAPERGS. V.F. Campos, E.R. 2
Komninou and P.M.M. de Leon are students of the Graduate Program in 3
Biotechnology at Universidade Federal de Pelotas and are supported by Brazilian 4
CAPES, whereas J.C. Deschamps and O.A. Dellagostin are research fellows of 5
CNPq. 6
7
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24
63
Table 1. In vitro embryo development of bovine oocytes in vitro fertilized with sperm subjected to 1
various transfection procedures. 2
3
Sperm transfection system Oocytes *Cleaved (%)
*Blastocysts (%)
**PCR positive (%)
Control 59 36 (61)a
11 (18.6) 0a
Naked exogenous DNA 60 37 (61)a
12 (20) 3 (8.1)b
Lipofectamine 2000 59 39 (66)a
13 (22) 4 (10.2)b
NanoFect 73 57 (78)a
22 (30.1) 26 (45.6)c
Halloysite Clay Nanotubes 64 54 (84)b
15 (23.4) 22 (40.7)c
4 *Rates of cleavage and blastocyst formation are expressed as a percentage of the initial number of 5 oocytes. 6 **Rates of PCR positivity are expressed as a percentage of the number of cleaved embryos. 7
a-cWithin a column, rates without a common superscript differed (P<0.05). 8
9
64
Figure Captions 1
2
Fig. 1. Motility and viability of bovine sperm (panels A and B, respectively) before and after 3
transfection procedures. Data are expressed as means ± SEM (N=3). 4
5
Con
trol
Nak
ed D
NA
Lipo
som
e
HCNs
Nan
ofec
t
0
20
40
60
80
Before DNA incubation
After DNA incubation
Mo
tile
sp
erm
(%
)
Con
trol
Nak
ed D
NA
Lipo
som
e
HCNs
Nan
ofec
t
0
20
40
60
80
Before DNA incubation
After DNA incubation
Via
ble
sp
erm
(%
)
A
B
6
65
Fig. 2. Examples of PCR analysis. PCR reactions were performed on snap-frozen bovine embryos. 1
Upper panel shows pEGFP-N1 fragment amplification and lower panel shows 1.715 satellite fragment 2
amplification. Lane 1 – positive control, Lane 2 – control embryo (without transfection). Lane 3 –naked 3
DNA group. Lane 4 – lipofection group. Lane 5 – Nanofect group. Lane 6 – halloysite clay nanotubes 4
group. NTC – no template control reaction. 5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
66
Fig. 3. Exogenous DNA quanfication in bovine sperm and embryos by real-time PCR. a) Plasmid 1
uptake by sperm cells from various transfection procedures. b) Transmission of plasmids to four-cell 2
embryos after various transfection procedures. Data are expressed as means ± SEM (N=3). 3
4
Con
trol
Nak
ed D
NA
Lipo
som
es
HCNs
Nan
ofec
t
0
50
100
150
200
a
b
a
c
cP
lasm
ids /
sp
erm
ato
zoo
n
Con
trol
Nak
ed D
NA
Lipo
som
es
HCNs
Nan
ofec
t
0
10
20
30
40
50
60
a
b
a
b
Pla
sm
ids /
sin
gle
4-c
ell
em
bry
o
A
B
5
6
7
8
9
67
1
2
3
4
5
6
7
4. CONCLUSÃO 8
9
Neste estudo foi possível concluir que: 10
11
1) O nanopolímero catiônico pode ser usado com sucesso para a transfecção 12
de DNA exógeno em espermatozóides bovinos sexados e não-sexados 13
sem nenhum efeito na motilidade e viabilidade espermática. 14
2) O nanopolímero catiônico e os nanotubos de haloisita incrementam tanto a 15
transfecção de DNA para os espermatozóides e a transmissão do 16
transgene para embriões bovinos sem nenhum efeito deletério ao 17
desenvolvimento embrionário. 18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
68
1
2
3
4
5
6
7
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