基于RCAS-TVA技术的哺乳动物模型(英文)-USB迷|专注于互联网分享

2024年5月22日发(作者：宁清卓)

动物学研究 2008，Jun. 29(3)：335—345 CN 53-1040/Q ISSN 0254-5853

Zoological Research DOI:10.3724/SP.J.1141.2008.03335

Mammalian Models Based on RCAS-TVA Technique

NIU Yi-dong

1,*

, LIANG Shu-long

(1. Laboratory Animal Center, Peking University People’s Hospital, Beijing 100044, China;

2. School of Basic Medical Sciences, Peking University, Beijing, 100083, China)

Abstract: The retroviral vector (RCAS) has been widely used in avian system to study development and diseases, but

is not suitable for mammals which do not produce the retrovirus receptor TVA. In this review, we trace the current uses of

RCAS-TVA approach in mammalian system with improved strategies, including generation of tv-a transgenic mice, use of

soluble TVA receptor and retroviral receptor-ligand fusion proteins, improvement of RCAS vectors, and compare a series

of mammalian models in variant studies of gene function, development, oncogenesis and gene therapy. All those studies

demonstrate that the RCAS-TVA based mammalian models are powerful tools for understanding the mechanisms and

target treating of human diseases.

Key words: RCAS vector; TVA; Specific promoter; Transgenic animal; Mammalian model

基于RCAS-TVA技术的哺乳动物模型

牛屹东

1,*

，梁蜀龙

（1．北京大学人民医院实验动物中心，北京 100044；2．北京大学基础医学院，北京 100083）

摘要：近年来，鸟类逆转录病毒载体（RCAS）及其受体（TVA）系统在哺乳动物转基因模型中得到广泛应用。

本文对转tv-a基因小鼠的制备、特异性启动子选择、RCAS载体的改进等方面进行综述，展示近来RCAS-TVA系

统在哺乳动物所取得的成果，并对RCAS-TVA基因转移技术的应用前景作一展望。

关键词：RCAS载体；TVA；特异性启动子；转基因动物；哺乳动物模型

中图分类号：Q812; Q78 文章标识码：A 文章编号：0254-5853-(2008)03-0335-011

Retroviruses are enveloped viruses possessing a

RNA genome, and replicate via a DNA intermediate.

They rely on the enzyme, reverse transcriptase, to

perform the reverse transcription of their genomes from

RNA into DNA, which can then be integrated into the

host's genome. The viruses then replicate as part of the

cell's DNA (/wiki/Retrovirus). So

they have the ability to introduce new genetic

information into the chromosomes of target cells, and

serve as vehicles for transfer of exogenes (Orsulic, 2002).

To date, retroviral vectors have been wildly developed to

study gene function and therapy, developmental

Received date：2008-01-17； Accepted date：2008-03-28

processes, oncogenesis, and so forth (Logan & Tabin,

1998; Hu & Pathak, 2000; Barton & Medzhitov, 2002;

Kawakami et al, 2003; Pao et al, 2003; Harpavat &

Cepko, 2006; Du & Li, 2007). Among those retroviruses,

the avian sarcoma-leukosis virus-A (ASLV-A)-derived

vector called RCAS (Replication Competent ASLV long

terminal repeat with Splice acceptor) is used most

extensively in avian system, because high titer viral

stocks can be harvested in avian cells without helper

components. RCAS vectors are derived from the SR-A

strain of Rous sarcoma virus (RSV) by deleting the src

oncogene with a multi-cloning site where exogenes can

Foundation item: Supported by Peking University People’s Hospital Research and Development Foundation (RDB2007-03)

收稿日期：2008-01-17；接受日期：2008-03-28

基金项目：北京大学人民医院研究与发展基金（RDB2007-03）

通讯作者（Corresponding author），Tel: 86-10-88325990, E-mail: niuyd07@

336 Zoological Research Vol. 29

be inserted (Hughes & Kosik, 1984; Hughes et al, 1987;

Petropoulos & Hughes, 1991; Boerkoel et al, 1993). The

multi-cloning site can stably accommodate inserts up to

2.5

kb (Fig. 1). Expression of the inserted genes can be

driven by either the viral long terminal repeat (LTR) or

an appropriate internal promoter (Petropoulos et al, 1992;

Du et al, 2006). A loss-of-function method, RCAS-RNAi

(RNA interference) technique, has been verified to be

efficient in “knocking down” the specific genes in

avian developing craniofacial tissues, the limb bud,

dorsal root ganglion, and the retina (Kawakami et al,

2003; Pekarik et al, 2003; Harpavat & Cepko, 2006).

Fig. 1 Constructs of the RSV and RCAS vector

The diagrams show the organization of viral DNA genomes and the

location of genes (gag, pol, env, and src) and long terminal repeat

(LTR). The src oncogene of RSV carries a splice acceptor (SA), which

is retained in RCAS. The src oncogene has been deleted and replaced

by a multi-cloning site in RCAS vector.

However, RCAS vectors can not be used in

mammalian system directly without any improvement on

the mammalian cells, which do not express the surface

receptors for virus entry and infection. TVA, a member

of the low-density-lipoprotein receptor family, is

encoded by the tv-a gene and acts as the receptor for

ASLV-A in avian cells (Bates et al, 1993; Young et al,

1993). mRNA transcribed from the tv-a gene is

alternatively spliced to produce at least two proteins, a

transmembrane and a GPI-anchored isoform (Bates et al,

1993). The mammalian cells are able to be infected and

allow for genome integration by ASLV-A or RCAS virus

if the cells are engineered to express TVA ectopically on

the surface, and both of the isoforms are sufficient to

permit infection of mammalian cells (Bates et al, 1993;

Young et al, 1993).

Compared with the general mammalian counterparts,

RCAS vectors can be constructed to encode all of the

proteins required for assembly of infectious particles in

addition to the transferred gene of interest, so they do not

require helper cells (Hughes et al, 1987; Petropoulos &

Hughes, 1991; Boerkoel et al, 1993). High-titer viral

stocks can be produced in avian cells (Himly et al, 1998;

Schaefer-Klein et al, 1998). Viral proteins are

inefficiently produced in mammalian cells, so the vectors

can not spread from the target animals and cell-to-cell

spreading within any individual is also prevented (Wills

et al, 1989; Berberich et al, 1990). The lack of viral

proteins also decreases the immune response by the host

(Pinto et al, 2000). Furthermore, the most specific

advantage of RCAS vectors is that multiple genes can be

transferred sequentially into the target cells of a single

transgenic animal (Federspiel et al, 1994; Holland et al,

1998; Murphy & Leavitt, 1999). This feature should be

attributed to the sufficient supplement of TVA receptor

which is not blocked by the poorly expressed viral

envelope protein in mammalian cells. Recent

experiments indicate that the RCAS vectors have the

ability to infect non-dividing mammalian cells, including

the primary neurons, although there is no direct evidence

(Hatziioannou & Goff, 2001; Katz et al, 2002; Greger et

al, 2004). The procedure to generate a RCAS-TVA based

model is shown in Fig. 2.

Some potential limitations for using the RCAS-TVA

system, however, should be given close attention. Target

cells, tissues and organs must express the receptor TVA.

Therefore, it is crucial to generate TVA transgenic

animals before RCAS infection. RCAS can only

accommodate an insert of less than 3

kb, but this

limitation can be partially overcome by using

pseudotyped vectors. The MLV (moloney murine

leukemia virus), carrying capacity of insert up to 6-7

kb,

has been efficiently pseudotyped with ASLV envelope

protein (Soneoka et al, 1995; Murphy & Leavitt, 1999).

But, integration site of viral DNA can not be controlled.

Efficiency of infection is dependent on the accessibility

of the organ and the proliferation rate of target cells.

Description of various advantages and limitations of

using the RCAS-TVA system has been reviewed in detail

by Orsulic (2002).

1 Transgenic mammals expressing TVA

molecules

1.1 Choice of specific promoters

The key element to produce tv-a transgenic animals

is the tissue-specific and the lineage-specific promoters

which decide specific expression of TVA in target cells,

tissues and organs. Therefore, a nucleotide fragment

No. 3 NIU Yi-dong et al: Mammalian Models Based on RCAS-TVA Technique 337

consisting of the tv-a cDNA and a proper promoter must

be constructed before RCAS infection. The specific

promoters currently used to drive expression of tv-a gene

in mammalian system are summarized in Tab. 1.

338 Zoological Research Vol. 29

Fig. 2 Schematic drawing of the RCAS-TVA technique in mammalian system

A: The target mammalian somatic cells are engineered to express TVA receptor under a tissue-specific promoter and therefore are

susceptible to virus infection. B: Avian cells are transfected with a plasmid encoding the replication-competent, avian viral vector RCAS

which contains the viral genes, gag, pol and env and a gene of interest (X gene). The viruses are produced in high titer and can infect

avian cells again through the TVA receptors on the surface of cells. C: The mammalian cells expressing TVA are infected by RCAS

vectors, and only the protein encoded by X gene is efficiently produced. Because very little viral proteins are produced, no infectious

RCAS are replicated in mammalian cells. Therefore, the TVA receptors can be used repeatedly with different vectors. The neighbor cells

can not be infected by RCAS because of their deficiency of TVA receptor. D: The target cells transfected with the genes of interest will

show different destinies, such as proliferating or dying.

Tab. 1 Summary of the target cells, tissues and organs expressing TVA and

promoters used to drive expression of tv-a in mammalian system

Target Promoter/contex

Bone

Brain

Hematopoietic cell

Liver

Lung

Mammary epithelial cell culture

Mammary gland

MEFs

Most or all tissues

Neural crest cell

Ovary

Pancreas

RK3E cell line

Skeletal muscle and heart

Vascular endothelium

BSP

GFAP

nestin

GP-Ibα

albumin

SPC

MACT

MMTV

β-actin

TRP2

β-actin , keratin

elastase I

CMV

α-actin

Tie2

Reference

Li et al, 2005

Holland & Varmus, 1998; Yamashita et al, 2006

Holland et al, 1998

Murphy & Leavitt, 1999

Lewis et al, 2005

Fisher et al, 1999

Phillips et al, 2006

Du et al, 2006

Pao et al, 2003

Federspiel et al, 1996

Fisher et al., 1999

Orsulic et al, 2002; Xing & Orsulic, 2005

Kruse et al, 1993; Lewis et al, 2003

Fu et al, 2005

Federspiel et al, 1994

Montaner et al, 2003

BSP, bone sialoprotein; CMV, cytomegalovirus; GFAP, glial fibrillary acidic protein; GP, glycoprotein;

MACT, mouse β-actin; MEFs, murine embryonic fibroblasts; MMTV, mouse mammary tumor virus; RK3E,

rat kidney epithelial cell line; SPC, surfactant protein-C; TRP2, tyrosinase-related protein-2.

Additionally, numerous studies have indicated that

some other promoters, including the ovary specific

promoter (OSP1) and the high-affinity folate receptors

promoter (HAFR) (Godwin et al, 1995; Goldsmith et al,

No. 3 NIU Yi-dong et al: Mammalian Models Based on RCAS-TVA Technique 339

1999), the modified rat probasin (rPB) promoters

(Furuhata et al, 2003), and the neuroactive peptide

cholecystokinin (CCK) promoter (Chhatwal et al, 2007),

are of potential value for tissue specific expression of the

tv-a gene.

1.2 Both isoforms of TVA molecule are sufficient for

acceptance of the RCAS vectors

As mentioned earlier, two isoforms, a

1999; Fisher et al, 1999).

However, the microinjection method has some

potential limitations: 1. limited success in producing

transgenic animals of larger species; 2. requirement of

special equipment for DNA microinjection and high

technical skills; 3. labor intensive. The new mean has

been developed by using sperm cells, including

spermatogonia, as the vehicle to deliver exogenous DNA

transmembrane and a GPI-linked one, have been

identified. In the current tv-a transgenic mice, the

GPI-anchored isoform is commonly used (Federspiel et

al, 1996; Holland et al, 1998; Du et al, 2006), but the

transmembrane isoform has also be used successfully in

the study of Murphy & Leavitt (1999). Therefore, both

isoforms can accept the RCAS vectors although the

physiological functions have not been determined.

1.3 Gene transfer methods

The most widely used method to generate transgenic

animals is to microinject foreign DNA into the

pronucleus of a fertilized egg. Pronuclear microinjection

is conceptually straightforward, although it demands

special equipment and technical skill, and has the

additional feature that any cloned DNA can be used

(Palmiter & Brinster, 1986). The primary mammalian

model based on the RCAS-TVA approach was developed

in transgenic mice expressing TVA specifically in muscle

cells. They were generated by microinjecting a

nucleotide fragment consisting of the tv-a cDNA and

chicken α-actin promoter fragment into fertilized mice

eggs (Federspiel et al, 1994). Subsequently, Holland et al.

generated mice expressing TVA on the surface of glial

cells by microinjection of Gtv-a transgene, which is a 2.2

kb fragment of the GFAP promoter driving expression of

the quail tv-a cDNA and a fragment from the mouse

protamine gene (MP-1) supplying an intron and signal

for polyadenylation (Holland et al, 1998, 2000). Using

the microinjection method, then, tv-a transgenic mice

were extensively created to accept RCAS vectors in

cancer models of mammary, ovary, pancreas, liver, lung,

brain, vascular endothelium, melanoma, and other cell

types (Korfhagen et al, 1990; Holland & Varmus, 1998;

Holland et al, 1998; Fisher et al, 1999; Orsulic et al,2002;

Montaner et al, 2003; Lewis et al, 2003, 2005; Pao et al,

2003; Fu et al, 2005; Du et al, 2006) and development

models, including neuron, hemapoietic cell lines, and

other organs (Doetsch et al, 1999; Murphy & Leavitt,

into oocytes, and is therefore called “sperm-mediated

gene transfer” (SMGT, Lavitrano et al, 1989). Based on

SMGT, transgenic rats (Hamra et al, 2002; Orwig et al,

2002), pigs (Honaramooz et al, 2002) and goats

(Honaramooz et al, 2003) have now been produced. As

the improved method of SMGT, testis-mediated gene

transfer (TMGT) has been demonstrated to be practical

in delivering foreign DNA directly into the interstitial

space of adult mammalian testes (Fig. 3A), and then the

exogenous DNA is transmitted to oocytes via fertilization

(Sato et al, 1999; Sato & Nakamura, 2004). Recently, He

et al (2006) further indicated that transgenic efficiency of

TMGT was very high in both F1 and F2 mice offspring

(41% and 37% respectively), and that TMGT was

suitable for creating transgenic animals. The TMGT

technique is very simple and convenient. A needle, a

plastic disposal syringe, and a dissecting microscope are

sufficient for delivery of DNA. The TMGT technique

opens a new perspective for generating tv-a transgenic

mammals (Fig. 3B), although it requires further

improvement.

More recently, Yang et al (2007) established a rapid

procedure for obtaining transgenic mice by directly

injecting exogenes into the ovaries of fertile mice, called

ovary mediated gene transfer (OMGT). After natural

fertilization, healthy transgenic mice were obtained, and

the introduced foreign gene was inherited by F1

offspring (64.9%) and transmitted to F2 progeny

(66.94%) stably. The foreign gene was found to be not

only integrated into the genome with a high frequency of

85.71% (multiple site versus single site insertions

analyzed by FISH), but also translated into a functional

protein and transferred to the next generation. Although

the procedure is somewhat more complicated than

TMGT, OMGT is still a useful technique with a much

higher success rate for creating transgenic mammals via

efficient and functional integration of the foreign gene

into the host genome and stable transmission of the

340 Zoological Research Vol. 29

Fig. 3 Schematic drawing of testis-mediated gene transfer (TMGT) method and retroviral gene delivery to mammals in vivo

A: Injection of solution containing tv-a gene driven by tissue-specific promoter and liposomes (in some cases) is performed at the corner of the

testis near the caput epididymis to a depth of 5-6

mm (Sato et al, 1999). B: The adult male mice carrying tv-a gene driven by a tissue-specific

promoter are mated with normal females，and the promoter-tv-a sequence will be delivered into zygotes by sperms. The offspring (F1

generations) are examined, and only those expressing TVA in specific tissues or organs are left for retroviral infection. Chicken DF-1 cells

transfected with RCAS vectors carrying genes of interest are propagated to obtain high titer viruses. Producer DF-1 cells, cell supernatants, or

concentrated viruses can be used to infect TVA-expressing mice. Tissue-specific infection can be achieved by direct injection of viruses or

virus-producing cells into an organ in which TVA is expressed

The anatomy atlas of testis is modified from the website

/Webanatomy/image_database/Reproductive/.

foreign gene to the offspring. Simplicity of procedure

and cost-effectiveness are the advantages of OMGT used

for tv-a transgenic animals in contrast to other traditional

methods, such as pronuclear microinjection.

2 RCAS vectors used for mammalian sys-

tem

2.1 Modified vectors for overcoming the limited in-

sertion size

No. 3 NIU Yi-dong et al: Mammalian Models Based on RCAS-TVA Technique 341

As mentioned earlier, RCAS can only accommodate

an insert of less than 3 kb. This may not be a significant

problem because most cDNAs studied are less than 2.5

kb (Fisher et al, 1999). This limitation can, however, be

overcome by using RCAS and ASLV-A Env pseudotyped

HIV and MLV vectors, respectively (Murphy & Leavitt,

1999; Lewis et al, 2001), even if more than 3

sequences must be inserted.

2.2 Improved vectors for infection of a broad range

of cell types

Although some reports have shown that RCAS can

infect non-dividing cells (Lu et al, 1999; Hatziioannou &

Goff, 2001), low efficiency suggests entrance of viral

DNA into nucleus depends on mitosis of host cells. To

overcome this limitation, Lewis et al (2001) succeeded in

using a pseudotyped replication-deficient HIV-1 based

lentiviral vector to infect non-dividing TVA positive cells.

However, there is no evidence that can clarify the

efficiency of this vector in vivo. Therefore, further

studies are required to improve the ability of RCAS

vectors into the genome of non-dividing host cells.

2.3 Vectors used in mammalian system

The RCAS family consists of a group of vectors for

variant demand. Actually, the current vectors used in

mammalian system, have an env gene from a murine

retrovirus instead of one from the ASLV. These vectors

are named as RCASBP, in which env gene is derived

from an amphotropic virus or ecotropic virus. The

properties of RCASBP vectors are summarized on the

website:

/hivdrp/RCAS/#table2.

3 Overview of current mammalian models

based on RCAS-TVA technique

The primary mammalian model based on

RCAS-TVA approach was developed in mice (Mus

musculus) by Federspiel et al (1994). This work opens a

new way to study development and oncogenesis, and

sheds light on models for tissue-specific gene therapy.

3.1 Use of RCAS-TVA based models to study

developmental processes

RCAS-TVA based method has been proven to be

useful in developmental studies in mammalian system.

Murphy & Leavitt (1999) used the GP-Ibα regulatory

sequences to achieve megakaryocyte-lineage of mice

restricted expression of TVA. They infected the cells

with RCAS-PURO (expresses puromycin-resistance

gene) and RCAS-AP (expresses human placental alkaline

phosphatase) in vitro and in vivo, then generated and

characterized a pure population of primary CD41-positi-

ve megakaryocyte progenitors. The in vitro study

indicated that IL-3 inhibits the development of mature

megakaryocytes. Doetsch et al (1999) infected SVZ

(subventricular zone) astrocytes of tv-a transgenic mice

with RCAS-AP in vivo, and the AP-positive cells were

examined and traced. They demonstrated that SVZ

astrocytes act as neural stem cells in normal brain. Study

of lung development has been reported using the

RCAS-TVA model (Fisher et al, 1999). Lung buds of the

SPC-tv-a transgenic mice were infected with different

RCAS viruses to study the effects on branching

morphology in vitro. To study bone development in vivo,

Li et al (2005) established the BSP-tv-a transgenic mice

which selectively expressed TVA in skeletal tissues.

After infecting with RCASBP-Cbfa1/Runx2, bone and

tooth formation was delayed. They validated this model

as a unique system for studying molecular events

associated with bone formation in vivo.

Dunn et al (2000, 2001)have infected the neural

precursor cells and the melanoblasts expressing TVA

driven by nestin and Dopachrome tautomerase promoter

(DCT) with RCAS-Wnt, RCAS-lacZ (β-galactosidase)

and RCAS-Tyr (tyrosinase) respectively in primary

culture and in utero. They demonstrated that the

RCAS-TVA method was useful to study the development

of neural systems. Recently, the RCAS-TVA system was

successfully adapted by Yamashita and colleagues to

study neurogenesis in vivo (Yamashita et al, 2006). They

traced maturation of neurons by infecting the GFAP tv-a

(Gtv-a) transgenic mice with RCAS-EGFP

(CAG-CAT-enhanced green fluorescent protein), and

indicated that SVZ-derived neuroblasts differentiated

into mature neurons in the post-stroke striatum.

3.2 Use of RCAS-TVA based models to study onco-

genesis

Currently, most models of tumors are traditionally

germ-line models constructed by transgenic or knockout

approaches. The major limitation of these models is that

the initiation and progression of carcinogenesis can not

be understood. However, the RCAS-TVA method

overcomes this limitation and allows investigation of the

carcinogenic potential of candidate oncogenes in somatic

342 Zoological Research Vol. 29

cells in vivo without creating individual transgenic lines

(Du & Li, 2007). To date, several oncogenes have been

studied in murine system using this technique. The

variant cancers or tumors and oncogenes studied are

summarized in Tab. 2.

Cancer is thought to be associated with multiple

genetic alterations. Microarray analysis of ovarian cancer

has demonstrated that oncogenesis of ovarian neoplasms

is controlled by many genes, and that changes in

expression of these genes correlate with malignancy

potential (Warrenfeltz et al, 2004). To study the effect of

multiple genes on carcinogenesis, the RCAS-TVA

system provides a flexible method to deliver several

genes simultaneously or sequentially. Holland et al (2000)

infected the Ntv-a transgenic mice with a combination of

DF-1 cells infected with and producing RCAS-Ras and

RCAS-Akt. They found that combination of activated

Ras and Akt induces high grade gliomas with the

histological features of human glioblastoma multiformes

(GBMs) although neither activated Ras nor Akt alone is

sufficient to induce GBM formation.

Orsulic et al (2002) isolated ovarian cells from TVA

transgenic mice deficient for p53, and infected the target

cells with RCAS-Myc, RCAS-Ras, and RCAS-Akt. Their

study showed that addition of any two of the oncogenes

Myc, Ras, and Akt were sufficient to induce ovarian

tumor formation when infected cells were injected into

the recipient mice at subcutaneous, intraperitoneal, or

ovarian sites. They demonstrated that the ovarian surface

epithelium is the precursor tissue for these ovarian

carcinomas, and that introduction of oncogenes causes

phenotypic changes in the ovarian surface epithelial

cells.

A mouse model for hepatocellular carcinoma was

generated by infecting tv-a transgenic wild-type and p53

null mice with RCAS-PyMT (Lewis et al, 2005). Tumors

were induced in both wild-type and p53 null mice, but

only in the mice lacking an intact p53 gene the resulting

tumors were poorly differentiated, invasive, and

metastatic to the lungs. This study demonstrates that

metastasis is dependent on both the oncogene and the

absence of p53.

3.3 Use of RCAS-TVA based models to study gene

function

The lost-of-function and “knock out” techniques

are robust and practical for studying gene function in

mammalian system. The retroviral vectors have been

validated to express short hairpin RNA (shRNA) under

the control of an RNA polymerase III promoter for the

purpose of inhibiting gene expression in a sequence-

specific manner (Brummelkamp et al, 2002; Hemann et

al, 2003; Rubinson et al, 2003).

To date, RNA interference (RNAi) technique has

been incorporated successfully with the RCAS-TVA

method to study gene function in avian development.

Bron et al (2004) knocked the neuropilin-1 (Nrp-1)

receptor in chick embryos using the RCAS-RNAi

technique. They found that Sema3A-induced growth

cone was inhibited in dorsal root ganglion (DRG)

neurons. This result demonstrated the functional

knockdown of Nrp-1. Harpava and Cepko (2006)

delivered hairpins mediating RNA interference to the

Tab. 2 Cancers/tumors and oncogenes studied in murine system by RCAS-TVA approach

Cancer/tumor Oncogene Target

Breast cancer

Liver cancer

Nervous system tumors

Astrocytoma

Glioblastoma

Oligoastrocytoma

Oligodendroglioma

tumors (PNETs)

Ovarian cancer Myc, Ras, Akt Ovarian cells in culture

Ovarian cancer cell lines and tumors with

defined genetic alterations

Pancreatic cancer PyMT, Myc Pancreas in vivo Lewis et al, 2003

Orsulic et al, 2002

Xing & Orsulic, 2005

Cre

PyMT, Neu

PyMT, Myc

Ras, Akt

PDGF-B

Mammary gland in vivo

Liver parenchyma in vivo

Brain in vivo

Brain in vivo, Primary brain cell cultures

Neural progenitor cells, brain in vivo

Reference

Fisher et al, 1999

Du et al, 2006

Lewis et al, 2005

Holland & Varmus, 1998

Holland et al, 2000

Holland & Varmus, 1998; Dai et al, 2001

Dai et al, 2001

Fults et al, 2002 Primitive neuroectodermal

Myc

PDGF-B, platelet derived growth factor-b chain.

No. 3 NIU Yi-dong et al: Mammalian Models Based on RCAS-TVA Technique 343

developing chick eye by RCAS viruses. They 'knocked

down' specific genes in infected areas of the retina. The

knock down persisted as the retina matured and could be

detected using in situ hybridization. Furthermore, the

amount of retinal tissue affected could be controlled by

manipulating the degree of infection.

In mammalian system, Bromberg-White et al (2004)

created a RCAS vector capable of expressing shRNA

that inhibits the expression of glyceraldehydes-3-phos-

phate dehydrogenase (GAPDH) gene, and reduces

GAPDH expression in cell line A375. They demonstrated

that RCAS vectors can be used to stably express shRNA

to inhibit gene expression in loss-of-function analyses of

specific genes in vitro as well as in vivo.

The RCASBP-Y vector has been modified to

incorporate “Gateway” site-specific recombination

cloning of genes into the viral construct, and will allow

for the efficient transfer and expression of cDNAs

required for functional genomic analyses in both avian

and mammalian model systems (Loftus et al, 2001).

3.4 Use of soluble TVA receptor, TVA-ligand bridge

proteins and RCAS system for gene therapy

RCAS system has been considered useful for gene

therapy of cancers (Orsulic, 2002; Xing & Orsulic, 2005).

However, gene therapy is dependant on the ability of

target cancer cells to accept the viral vectors carrying

therapeautic or suicide genes. Many TVA receptor

transgenic models have been generated in mammalian

system to accept RCAS vectors, simultaneously new

methods are developed. Several studies have indicated

that the membrane TVA receptor is not an absolute

requirement for virus infection, and RCAS vectors linked

with a soluble TVA can be delivered into

receptor-deficient cells (Snitkovsky & Young, 1998;

Damico & Bates, 2000; Contreras-Alcantara et al, 2006).

The viral receptor function of TVA is determined by a

40-residue, cysteine-rich motif called the LDL-A module,

which is highly homologous to the human low-density

lipoprotein receptor (LDLR) ligand-binding repeats

(LDL-A modules). It has been demonstrated that the

LDL-A module of TVA is necessary and sufficient to

mediate efficient EnvA binding and ASLV-A infection

(Rong & Bates, 1995). Therefore, the soluble TVA

receptor is an ideal candidate for transferring RCAS to

target cells deficient in membrane TVA.

Additionally, several proteins consisting of a TVA

receptor-ligand fusion structure have been developed to

serve as bifunctional bridge to surface receptors of target

cells and RCAS vectors (Fig. 4). The bridge proteins

contain the extracellular domain of TVA and a peptide

which can bind to surface receptors of target cells

(Orsulic, 2002). Several authors have succeeded in

infecting the mammalian cells expressing cognate

cellular receptors using the bridge proteins consisting of

the domain of epidermal growth factor (EGF), vascular

endothelial growth factor (VEGF), or heregulin (Boerger

et al, 1999; Snitkovsky et al, 2000, 2001; Snitkovsky &

Young, 1998, 2002). This method provides a flexible

way to target entry of RCAS vectors to mammalian cells.

Fig. 4 Delivery of RCAS vectors into target mammalian cells

by retroviral receptor-ligand fusion proteins

The fusion protein is comprised of the extracellular domain of TVA

fused with a ligand protein which permits it to bind to RCAS viral ENV

and to cell surface receptor respectively.

Hu et al (2007) investigated for the first time the

characteristics of RCAS as an alternative vector system

for transduction of hematopoietic stem and progenitor

cells. The new vectors were modified by replacing the

avian env gene with the gene encoding amphotropic or

ecotropic murine Env protein, which allows RCAS

vectors to infect mammalian cells efficiently (Barsov &

Hughes, 1996; Barsov et al, 2001). They used nonhuman

primate autologous transplantation models to test

whether the RCAS vectors can efficiently transduce

rhesus macaque CD34

hematopoietic stem and

progenitor cells. This study showed that RCAS vectors

could efficiently and stably transduce the CD34

hematopoietic progenitor cells with an efficiency of

transduction of up to 34% in vitro, and that highly

polyclonal hematopoietic reconstitution in myeloid and

lymphoid lineages was observed up to 18 months

post-transplantation in animals transplanted with RCAS

vector-transduced autologous CD34

cells. Hu et al

344 Zoological Research Vol. 29

(2007) indicated that the RCAS system should be

explored and further optimized for gene therapy

applications targeting hematopoietic stem and progenitor

cells.

4 Conclusion and future research

This review shows that the RCAS-TVA based

technique is very useful and valid in various areas,

including basic biology, medicine and clinical research.

To date, a large number of RCAS vectors (and ancillary

tools, including soluble receptors, receptor-ligand fusion

proteins, mammalian cell lines expressing receptors, and

tv-a transgenic mice) have been developed for

mammalian system, and the number of mammalian

models (especially the mouse models) is constantly

increasing. These models can be used for a number of

different study purposes: gene function, development,

carcinogenesis and gene therapy.

Bioreactor is also a novel potential for the use of

RCAS-TVA system in the future. Pronuclear

microinjection is the major method today for the

production of transgenic animal bioreactor, but repeated

operation is inevitable to create different transgenic

animals producing different bioactive proteins. The

RCAS-TVA technique provides a convenient and flexible

way to produce a variety of biological products in one

transgenic animal, in which different target genes can be

introduced to the tissues or organs expressing TVA

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